Methods and compositions for targeted drug delivery

ABSTRACT

Disclosed are Drug Delivery Molecules (DDMs) which both facilitate functional imaging, as by PET, MRI or SPECT, and create a biological effect and methods of their use. These DDMs which are variously designed to target specific receptors, internalized and then function biologically, as for purposes of cell destruction or therapy.

This application is a continuation-in-part of Ser. No. 10/238,755, filedSep. 9, 2002, which claims priority from U.S. Provisional ApplicationSer. No. 60/318,270, filed on Sep. 7, 2001, and which is also acontinuation-in-part of U.S. Ser. No. 09/936,094, filed Sep. 7, 2001,which is a continuation of international application PCT/US2000/06001,filed Mar. 8, 2000, the disclosures of all of which are incorporatedherein by reference.

FIELD OF INVENTION

The present invention is directed to drug delivery molecules that have atargeting moiety, a routing moiety and a bioactive molecule (BAM),wherein the targeting moiety significantly binds to a receptor on thesurface of a cell. Binding of the targeting moiety to the receptor doesnot elicit a significant agonistic effect; however, it results in anuptake of the drug delivery molecule into the cell. The routing moietyis suitably coupled to the bioactive molecule, and the targeting moietymay be coupled either to the routing moiety or to the bioactivemolecule. The bioactive molecule may be one that is cell-destructive, orit may be one that is useful in gene therapy. Methods and compositionsdrawn to particular target cells, uses, receptors, and drug deliverymolecules are described in co-pending application U.S. Ser. No.09/428,675 filed Oct. 27, 1999, which is incorporated herein byreference, and Ser. No. 09/936,094. Various alternative target cells,uses, receptors, and drug delivery molecules are contemplated beyondthese disclosures. In general, all cells that express somatostatinreceptor type 2 (i.e. SSR-2) are considered to be particularly suitabletarget cells for use in conjunction with the teachings presented herein.This definition is intended to expressly include regular (i.e. healthy)cells as well as diseased cells expressing and presenting the SSR-2.Healthy cells which express present SSR-2 include epithelial cells,smooth muscle cells, neuronal and neuroendocrine cells. Of these,certain epithelial cells that are particularly contemplated are suchcells of the gastrointestinal (GI) tract, liver epithelia (parenchyma),and bile duct epithelia. As used herein, the term “gastro-intestinaltract” refers to the section of the alimentary tract located aborallyfrom the esophageal/gastral fusion, i.e., the region beginning with thefundus of the stomach and ending with the anal portion of the rectum.Also, included under this definition are the abdominal organs, i.e.liver and pancreas, that are connected to the alimentary tract by largesecretory ducts. Additional suitable target cells in the GI tractinclude epithelial cells lining the inner surface of the gut, andespecially those lying in the strata undergoing rapid proliferation. Incontrast, fibroblastic cells of the stroma and healthy endothelial cellsof blood vessels are not considered to be candidates.

Especially contemplated neuroendocrine cells include glugagon-producingislet cells of the endocrine pancreas and pituitary cells havingneuroendocrine characteristics. Particularly contemplated neuronal cellsinclude peripheral somatostatinergic neurons, in particular in thecardiac atrium, and cells of the CNS (central nervous system) and PNS(peripheral nervous system) that express/present the SSR-2.

BACKGROUND OF THE INVENTION

Upon biosynthesis at the ribosomal complex, proteins undergo a series ofediting steps through which proper folding and/or proper modification(e.g. by attachment of carbohydrate chains, lipid chains, or byphosphorylation) are ascertained. The editing steps occur in a stepwisemanner on the surface, or within, highly specialized vesicularstructures that are part of a highly dynamic network of vesicles withinthe cytoplasm, termed endoplasmic reticulum. In order to guide proteinsthrough the editing processes, unique elements within the proteinstructure have evolved that act as sorting signals, by facilitatinginsertion of a given protein into membranes (a well-recognized functionof attached lipid or carbohydrate residues), by defining interactionwith so-called chaperones (transport molecules that move a given proteininto or from a subcellular environment), or by a combination of suchmechanisms. In essence, specific amino acid sequences acting as sortingsignals match a given protein to its final destination where it is toexert its function, e.g. the plasma membrane for a surface receptor, thenucleus for a transcription factor, the mitochondrium for an enzymeparticipating in the respiratory chain. Many principles of sorting havebeen elucidated by the seminal studies of Blobel who was awarded theNobel Prize for Medicine and Physiology in 2001. A large number ofresearch groups have assembled the current knowledge about sortingsignals for individual organelles (see Trends in Biochem Sci 16,478-481, 1991 for an overview over nuclear translocation sequences; seeHartl and Neupert, Science 247, 930-938, 1990; Pfanner et al., Annu Rev.Cell Dev. Biol. 13, 25-51, 1997 for review of mitochondrialtranslocation sequences). The details of sorting are highly complex anddiffer with cellular environments, and most aspects of sorting,especially how multiple sub-elements of a sorting signal function, orhow multiple signals for different destinations within a proteinstructure are assigned their order and priority of processing, arecurrently not understood.

In particular, the details of mitochondrial import of proteins are stillunclear. The inner space of mitochondria is surrounded by two distinctmembranes. Within each membrane, a designated protein complex acts asgatekeeper for macromolecules crossing the membrane. There is no“consensus sequence” of mitochondrial import signals that could act asreference for studies on structure-function-relationship. Mostmitochondrial import signals are of higher complexity and are encoded byrelatively long peptide sequences. After import, the signal sequence ofnaturally occurring proteins is cleaved off by the translocase. Therecent crystal structure analysis of the enzyme importing macromoleculesinto the inner space of mitochondria has revealed some structuralinsight about the structural requirements on import sequences. Repeatsof short helical structure are facilitating the translocation stepproper, whereas the protein region encoding the import signal has toundergo a conformational change into a more unfolded structure to permitproteolytic cleavage off the imported protein. The sum of the individualprocesses of guided intracellular movements of a given macromoleculefrom one environment to another is often collectively referred to as“trafficking”.

The invention contemplates to reduce drug toxicity and to increase drugefficacy by imparting the trafficking pathways of endogenousmacromolecules upon therapeutic and imaging drugs designed to exerttheir function within a specific subcellular environment.

For the sake of clarity, definitions are given as follows (see also FIG.2 and the legend to FIG. 2 in co-pending application U.S. Ser. No.09/428,675 filed Oct. 27, 1999): A “presentation molecule” encompassesan extracellular targeting moiety, connected to an intracellulartargeting moiety, termed “routing moiety”, that is carrying a“bio-active molecule”, or BAM.

The extracellular targeting moiety acts as a ligand (preferably anon-agonist ligand) for a transmembrane receptor that participates ininternalization (a process wherein a membrane protein leaves themembrane environment, releases bound ligands to the intracellular space,and eventually returns to the membrane environment). The “routingmoiety” participates in intracellular trafficking and delivers the drugmolecule to a pre-determined subcellular environment. The “bioactivemolecule” is the pharmacologically most active element in thecomposition and exerts the therapeutic effect, and/or the imagingfunction. In the case of a dual modality PET(SPECT)/BNCT drug, therouting moiety and the bio-active molecule are fully integrated,generating a unique drug delivery molecule (DDM). “Subcellularenvironment” is the collective term for all cellular organelles (such asnucleus, mitochondria, etc.), vesicular networks (such as theendoplasmatic reticulum with the Golgi apparatus), dynamically changingvesicular complexes (such as lysosomes and vesicles encountered intrafficking), and the cytoplasmic space between organelles and vesicularelements.

The use of ¹⁰B which when subjected to epithermal neutrons decays tolithium-7 and an alpha particle is well known and has been suggestedpreviously for destruction of cancerous cells, (see Inhibition of HumanPancreatic Cancer Growth in Nude Mice by Boron Neutron Capture Therapy,Hyyanagie, et al., British Journal of Cancer, 75 (5), 660-665 (1997). Itis frequently referred to by the acronym BNCT. The potential efficacy ofBNCT for malignant glioma is discussed in Boron Neutron Capture Therapy:Implications of Neutron Beam and Boron Compound Characteristics, F. J.Wheeler, et al., Med. Phys. 26 (7), 1237-1244 (July, 1999). Two articlesby Y. Imahori, et al. discuss PET based BNCT using boron compoundlabeled with ¹⁸F, see Clinical Cancer Research, 4, 1825-1841 (August,1998). U.S. patents relating to the use of BNCT include U.S. Pat. Nos.6,074,625, 6,248,305 and 5,846,741.

In essence, BNCT employs a stable non-radioactive isotope such asboron-10 (¹⁰B) which upon capturing a thermal neutron causes a fissionreaction. In the ¹⁰B fission, the resulting alpha and lithium particleshave high energy, LET and RBE and travel less than 10 microns in tissue.As a result, selective tumor-cell killing is provided. An external beamof epithermal neutrons, with an energy maximum between 100 and 1000 eV,is employed to treat the ¹⁰B -loaded tissues by focusing upon a specificregion in the body where the treatment is desired, and thus avoidingactivation of the boron isotope that may have been retained in otherparts of the body which may also contain the receptors being targeted.Details of reactor design and neutron beam validation are given inWheeler et al., Med. Phys. 26,1237-1244,1999. Lee et al. in Med. Phys.27, 192-202, 2000, describe a modified accelerator that is portable,more economical, and could make BNCT available for widespread hospitaluse. Alburger et al. in Med. Phys. 25, 1735-1738, 1998, have developedhighly sensitive phantoms for thermal neutron depth profiling that canbe used for validation of neutron beams obtained from accelerator-basedBNCT facilities. In absolute numbers, a ¹⁰B content of 5-30 ppm in tumorcells is necessary to cross the threshold for effective BNCT,corresponding to a number of about 10⁹ ¹⁰B atoms distributed uniformlythroughout a tumor cell (Fairchild and Bond, Int. J. Radiat. Oncol.Biol. Phys. 11, 831-840, 1985). The desirable therapeutic range between5 and 30 microgram/g tumor tissue (Coderre and Morris, Radiat. Res. 151,1-18, 1999). Subsequent calculation of tissue dosimetry has beenconventionally accomplished by Monte Carlo simulations of celldestruction using estimated intracellular and extracellularconcentrations of the radiopharmaceutical (Kobayashi and Kanda,Radiation Research 91, 77-94, 1982). The accuracy of such estimationscan be substantially improved by utilization of a boron compound that isaccumulating, and detectable by a second imaging modality, in apre-determined cellular microenvironment, preferably the nucleus whereneutron capture will exert the best therapeutic effect.

Monitoring and imaging of gene expression upon gene therapy has becomean important procedure for validation of experimental gene therapyregimens. Most gene repair efforts have to be targeted to a specificorgan, such as the liver or intestinal epithelia. Both temporal andspatial parameters of gene expression are to be assessed in livingorganisms in real-time mode to evaluate the effectiveness of the chosengene delivery procedure. A variety of imaging techniques has beentested. Weissleder et al. (Nature Medicine 6, 351-355, 2000) describe anexperimental procedure wherein a tumor made to overexpress an engineeredversion of the transferrin receptor could be visualized in vivo bytransferrin-linked paramagnetic particles detected by MRI. However, theincrease of paramagnetic particle uptake was only 2.5-fold compared totumors not expressing the engineered receptor gene, and it was concludedby Weissleder et al. that temporal resolution was limited, and probedetection sensitivity was several orders of magnitude lower than inalternative imaging modalities, such as optical imaging and imaging ofnuclear isotopes.

As alternative imaging modality, PET scanning has been testedsuccessfully. Certain isotopes emit positively charged particles of amass close to zero (positrons) that otherwise have the wave propertiesof negatively charged electrons. If a positron and an electron collide,each particle undergoes conversion into a gamma ray of 511 keV energy;since both gamma rays are emitted into opposite directions at an angleof 180 degrees, it is feasible to scan such conversion events ascoinciding gamma rays in paired detectors using e.g. lutetiumoxyorthosilicate as scintillation detection material, while eliminatingthose gamma rays that do not coincide. Details of current PETinstrumentation are described in Fahey, Radiol. Clinics North Am 39,919-929, 2001.

Because of high cost in running dedicated PET systems, a less expensiveimaging modality (single photon emission computed tomography, SPECT,also abbreviated SPET, for single photon emission tomography) hasrecently gained popularity whereby a gamma ray in the energy range of 30to 300 keV energy is emitted and detected by a modified dual-head, ormultiple head, gamma camera system. SPECT imaging can be performed withisotopes of longer half-life than those used in PET, such as ¹¹¹In or^(99m)Tc, that are well-characterized in nuclear medicine and can beshipped from dedicated radiochemistry facilities. SPECT imaging ofleukocytes labeled with ¹¹¹In or ^(99m)Tc has been validated as “goldstandard” in detection of occult infectious and inflammatory sites(Renken et al, Eur. J. Nucl. Med 28, 241-252, 2001). While the use ofconventional gamma camera technology is somewhat of an advantage forimage acquisition, conventional SPECT does not employ electroniccollimation of incoming gamma rays (Shao et al, Phys. Med. Biol.42,1965-1970, 1997), and thus is estimated to have lower sensitivitythan PET by at least one order of magnitude. Multiple head detectionsystems and advanced image construction software are critical to achieveoptimal imaging. SPECT imaging has been found to be a safe andcost-effective method with advantages over CT and other imaging methodsin diagnosis and management of lung cancer patients (Goldsmith andKostakoglu, Radiol. Clinics North Am. 38, 511-524, 2000). Details ofcurrent SPECT instrumentation, especially novel useful SPECT/PET hybriddetection systems, are described in Fahey, Radiol. Clinics North Am 39,919-929, 2001.

Increasing the concentration and limiting the source of SPECT in adefined microenvironment within a target cell by pharmacological meanswould be a significant improvement, both for image resolution and forradiation planning by other modalities such as BNCT. It is a particulardisadvantage and source of error if equal distribution of a radiationsource across all cell compartments has to be assumed, rather thanmeasuring it directly in the microenvironment that is important for theintended radiation therapy.

It has been proposed that SPECT and PET are useful to perform imaging inmouse models, with a resolution of about 1 to 2 mm and a signalcollection time in the range of minutes (reviewed by Weissleder, NatureReviews in Cancer 2, 2002); a mouse PET system to permit 1 mm resolutionhas been reported as in development by Hershman et al (J. NeuroscienceRes. 59, 699-705, 2000). Alternative imaging modalities include the useof fluorescent reagents (Honigman et al, Mol. Therap. 4, 239 -249, 2001)and Yang et al. Proc Natl Acad Sci 98, 2616-2621, 2001); the clinicaluse is still limited, because non-invasive detection is limited topathological processes close to the surface, with a maximal depth of 10cm in fluorescence-mediated tomography (Weissleder 2002).

Ray et al. have published a synopsis and a review about the applicationof PET to monitor gene therapy (see Table 1 in their publication in Sem.Nucl. Med. 31, 312-320, 2001). The best characterized model is by way ofdelivering a gene from Herpes Simplex virus encoding a thymidine kinasethat will accept as substrate synthetic derivatives of uracil (e.g.2′¹⁸F-2′-deoxy-1-beta-D-arabinofuranosyl-5¹²⁵I-uracil) and guanosine(e.g. ¹⁸F-ganciclovir or ¹⁸F-penciclovir), whereas the naturallyoccurring thymidine kinase does not. The level of gene expression to betested is proportional to the amount of phosphorylated uracil derivativeor guanosine derivative that is retained intracellularly uponphosphorylation and can be detected by PET scan. Similarly, the geneencoding somatostatin receptor type 2 has been delivered to cells topermit imaging by PET (Rogers et al., Q J Nucl Med 44, 208-223, 2000).This approach is still bound to the limitations of having anextracellular ligand contact a target of unknown and possibly lowsurface density, and cannot discriminate between cells naturallyexpressing the somatostatin receptor and those targeted successfully bygene therapy. Neuroendocrine tumors and gastrointestinal tissues expresssomatostatin type 2 receptor naturally; a tangible benefit for imagingby raising the level of somatostatin receptor further may apply to onlya small subset of tumors. Furthermore, gene therapy to the liver may notbe reportable at all. The Herpes virus thymidine kinase approach is farsuperior, because a detectable artificial intracellular substrate isenzymatically enriched only in cells that express the transgene atsufficiently high density. Still, it would be a significant improvementif the administration and overexpression of a viral enzyme interferingwith energy metabolism and DNA synthesis could be avoided.

Mitochondria play a major role in the metabolism of eukaryotic cells andcontrol pathological processes in disease and aging. For example,impaired mitochondrial function has a direct impact on ATP synthesis,regulation of intracellular Ca⁺⁺ homeostasis, generation of freeradicals, and execution of programmed cell death pathways. It istherefore of great interest to target pharmaceutical compositions tomitochondria, for the purpose of monitoring physiological arepathological processes, or to deliver therapeutic drugs (see Murphy,Trend in Biotechnol. 15, 326-330, 1997). Specifically, tumor cells arecharacterized by a higher cell membrane potential and also a highermitochondrial membrane potential (Chen, Annu Rev. Cell Biol 4, 155-181,1988), permitting compounds like lipophilic cations to accumulate with acertain selectively in tumor cell mitochondria. The disruptive effect oflipophilic cations may be exerted by increase of the proton permeabilityof the inner membrane and inhibition of respiration (Azzone et al, Curr.Topics Bioenerg. 13, 1-77, 1984), or by more specific inhibitory effectse.g. on mitochondrial transcription. Lipophilic cations have been usedas carriers to deliver the cytotoxic compound cisplatin to tumor cells(Steliou, U.S. Pat. No. 6,316,652). Furthermore, cation conjugates havebeen designed that combine to form a toxic product once inside thecancer cell mitochondrium (Rideout, Cancer Invest 12, 189-202, 1994).

Zhang and Haugland (U.S. Pat. No. 5,686,261) have disclosed fluorescentsubstituted 3′-6′-diaminoxanthenes which selectively localize inmitochondria and are retained after fixation, permeabilization, and celldeath. No pharmaceutical applications are contemplated. The use offull-length bovine heart mitochondrial protein DNA sequence is suggestedfor creating a fusion gene with a yeast gene of interest, which uponexpression will be imported into mitochondria. Herrnstadt et al. (U.S.Pat. No. 6,171,859) have disclosed methods and compositions to destroymitochondria with defective cytochrome c oxidase in patients withAlzheimer's disease by way of a toxin conjugated to targeting moleculewhich is a lipophilic cation. The toxin may be a small-molecule agent oran antisense oligonucleotide. Alternatively, an imaging ligand (e.g.radioisotope suitable for PET or SPECT) may be coupled to the targetingmolecule for in vivo imaging of defective mitochondria which willaccumulate the imaging drug through increased membrane potential andincreased levels of negatively charged phospholipids.

Because tumor cells inside of a tumor are often without significantoxygen supply, the mitochondrial membrane potential is not defined byrespiration, and thus may not be optimal for uptake of lipophiliccations. Milder forms of oxygen deprivation in the periphery of tumorsmay elicit a stress response leading to the observed hyperpolarizationof cancer cell mitochondrial and plasma membranes. Efficaciouslipophilic cation uptake is thus limited to a small portion of the tumorcells. Another limitation is the access of lipophilic cations tomitochondria in normal cells which represent a pool vastly in excessover cancer cell mitochondria. Alternative modes of mitochondrialtargeting are very desirable.

The use of mitochondrial import sequences instead of lipophilic cationsmight improve delivery of a drug to a larger population of mitochondriain a tumor, but would at the same time target mitochondria in non-tumorcells as well, thus losing a critical element of selectivity. Again,alternative modes of mitochondrial targeting are very desirable.

Somatostatin receptor ligands have been used to image cells byvisualizing the level of somatostatin receptor present on the surface ofa cell in a disease condition. Specific radiolabeled somatostatinanalogs are disclosed in EP 607103 to visualize somatostatin receptor inprimary tumors, tumor metastases, cells affected by inflammatory andautoimmune disorders, tuberculosis, and cells in organ rejection aftertransplantation. The use of somatostatin analogs for imagingsomatostatin receptor positive cells and tissues, in particular tumors,metastases, inflammatory disorders and autoimmune disorders, is furtherdisclosed in WO 97/01579. U.S. Pat. No. 5,976,496 discloses the use ofsomatostatin analogs to image atherosclerotic plaque, in particularnon-critically stenotic plaque and unstable atherosclerotic plaque.

Neither of the references suggests that somatostatin analogs may be usedto image angiogenesis by way of a tripartite molecule comprising anon-agonist targeting moiety, an intracellular routing moiety, and aradionucleid linked to the routing moiety. In the current invention, thesomatostatin analog is not derivatized to carry a radionucleid. Neitherof the references suggests using the somatostatin receptor as port ofentry, translocating the imaging drug to a pre-determined subcellularmicroenvironment by way of sorting signals, and attaching a radiolabelto a molecular target other than a somatostatin receptor.

SUMMARY OF THE INVENTION

In one aspect of the invention, drug delivery molecules which includetargeting and routing moieties, may be employed in situ for guided celldestruction. It is especially contemplated that the BAM is labeled witha label to be detected by an imaging method and carries a secondcell-destructive moiety, e.g. one that will selectively destroyparticular cells, as by being activated by the application of focusedenergy.

In a more particular aspect, it is contemplated that the drug deliverymolecule (DDM) is labeled with a radionuclide suitable for PET (positronemission tomography) or for SPECT (single photon emission tomography)and includes a boron compound; this combination allows detection of thedrug delivery molecule at its location in the body by PET(SPECT) andthereafter the destruction of the molecules' immediate cellularenvironment by the emission of radiation after capture by boron of slowneutrons which render it unstable and subject to radioactive decay.

Other examples of use of DDM include real-time monitoring of drugresistance in clinical tumors and the functional imaging of biologicalprocesses in mitochondria.

In one particular aspect, the invention provides a drug deliverymolecule which comprises a bioactive molecule (BAM) of a nature toeffect cell-destruction, a targeting moiety that does not activate areceptor to which it links, a routing sequence for causing delivery toan intracellular compartment in a cell having said receptor, and a labeluseful for imaging by Positron Emission Tomography (PET) or SinglePhoton Emission Tomography (SPECT).

In another particular aspect, the invention provides a drug deliverymolecule which comprises a bioactive molecule (BAM), a peptide targetingmoiety that does not activate a receptor to which it links, a routingsequence for causing delivery to an intracellular compartment in a cellhaving said receptor and a label useful for imaging by Positron EmissionTomography (PET) or Single Photon Emission Tomography (SPECT).

In another aspect of the invention, drug delivery molecules can be usedto target healthy and diseased cells, and to particularly effect genetherapy by import of appropriate bioactive molecules. As used herein,the term “gene therapy” refers to any modification in expression and/orinformation encoded in a nucleic acid. For example, modification ofexpression includes up-, and down regulation of transcription and/ortranslation, change in chemical stability of mRNA, degree ofpolyadenylation, recombinant expression of homologous and/or hetrologousnucleic acids, etc. Modification of information especially includesdeletion, addition, transition and transversion, or recombination ofnucleic acid with nucleic acid of a target cell. More particularly inthis aspect, common genetic liver disorders may be addressed by means ofgene therapy to correct, for example, hepatic acute porphyria (seereview Grandchamp, J. Gastroenterol. Hepatol. 11, 1046-52 (1996), inbornerrors of bile acid biosynthesis and transport (see review Balistreri,Gastroenterol. Clin. North. Am 28, 145-72 (1999), genetic disorders ofcopper and iron transport (see review Cox, Brit. Med. Bull. 55, 544-545,(1999), and Thalassemia and Hemophilia (see review Cahill and Colvin,Postgraduate Med. J. 73, 201-206 (1996). Gene therapy of such diseasesmay be performed by employing nucleic acid anti-sense or expressionconstructs as the BAM, however, various alternative BAMs may includerepressors, activators, etc. Where such a drug delivery moleculecomprises repressors, activators, nucleic acid anti-sense or expressionconstructs as the bioactive molecule, it should also be appreciated thatthe drug delivery molecule may be employed to modulate gene expression,for example, in liver injury and liver repair (Tracy and Fox, Semin.Pediatr. Surg. 5, 175-181 (1996). In addition to the treatment of liverdiseases by gene therapy in this manner, treatment of genetic diseasesin gastrointestinal epithelia, for example, vitamin B₁₂- or vitamin Dmalabsorption, gluten hypersensitivity, sprue, etc. is alsocontemplated.

In a further aspect of the invention, it is contemplated that the drugdelivery molecules may also be employed to assess the gene function ofgenomic or other nucleic acid material in vivo. For example, the drugdelivery molecule may have an EST (expressed sequence tag) expressioncassette, and such drug delivery molecules are delivered in vivo to anorganism. SSR-2 expressing cells will subsequently incorporate andexpress the EST, and a potential impact on the SSR-2 expressing cell maybe observed or a new protein may be purified. Where the EST includes aregulatory element, up and down regulation of genes in SSR-2-expressingcells may be monitored or detected. An alternative procedure to detectthe successful expression of the protein encoded by the EST-tagged geneintroduced into the target cell is monitoring of a SPECT signalgenerated by a small-molecule ligand binding to the EST. This procedureis applicable in vivo and is non-invasive, avoiding the need forrepeated biopsies which is particularly advantageous in the case ofliver monitoring, because of the known risk of bleeding.

Another example is targeted delivery of a coding sequence for the CFTRgene, combined with a chlorine isotope used to monitor restoration ofthe physiological clorine channel activity after gene therapy, or asmall-molecule affinity ligand for the segment of the CFTR protein to beexpressed by way of the gene therapy construct introduced. This would bein contrast to the strategy favored by Brown et al (J Bioenerg Biomembr29, 491-502, 1997) who proposed to influence protein folding by smallmolecule binding.

In a still further aspect of the invention, it is contemplated that theBAM in the drug delivery molecule may be employed to import peptides orother small molecules into a target cell, for example, to complement anintracellular biochemical or structural function of a cell, or tointerrupt specific cytokines and restore gastrointestinal function inMorbus Crohn and Colitis ulcerosa. For example, restenosis of arteriesafter surgery may be treated by employing a drug delivery moleculehaving a BAM which is a cell cycle interfering agent, or which isinhibitory to a growth factor causing elimination of the growth factorreceptor function).

All compositions and methods presented herein are considered suitablefor use in human and veterinarian diagnosis and treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Targeting of Alternative Surface Receptors

While it is generally preferred that the targeted receptors are SSR-2receptors, targeting of various alternative receptors is alsocontemplated, e.g. SSR-3 and SSR-5. For example, the SSR-3 receptor isparticularly prevalent in microvascular endothelia of Kaposi Sarcoma,and the SSR-5 receptor is particularly prevalent in neuroendocrinepancreatic beta cells (e.g. an advantageous target for restoration ofbeta cell function). Consequently, depending upon the indication beingtreated, the targeting moiety of the drug delivery molecule may be aligand that will specifically bind to one of SSR-2, SSR-3 or SSR-5.Preferred targeting moieties specific for SSR-3 and SSR-5 includemolecules that are either non-agonists or antagonists. Other alternativereceptors that may be targeted include the E-selectin membraneglycoprotein and the Duffy antigen/chemokine receptor, as well as theuPAR receptor, the vitronectin receptor (Åü

3), the EGF receptor, the CD40, CD36, and the CD11b surface antigen.

When the SSR-2 is to be targeted, all antagonists selective for theSSR-2 are suitable as targeting moieties. Although peptides arepreferred, pseudopeptides and nonpeptidic selective ligands areconsidered equivalents. Alternative targeting moieties include syntheticand/or natural ligands that specifically bind to a particular otherreceptor being targeted. For example, it is specifically contemplatedthat non- agonist ligands for SSR-2 may be linked to a BAM which isdesigned to complement known genetic defects in cystic fibrosis. Aparticularly preferred embodiment is a non-viral delivery vectorcomprising a non-agonist ligand for a surface receptor ofgastrointestinal epithelia connected to a bicistronic promoter elementthat controls expression of the therapeutic CFTR sequence and of amonitoring sequence encoding a unique EST wherein the EST is engineeredto be plasma membrane-associated and detectable by a radiolabeled plantor insect hormone, or an analog thereof.

With respect to the BAM, in an application designed forcell-destruction, it is especially contemplated that such may be aninherently toxic molecule, e.g. a peptide toxic to endothelial cells ora toxic lipid ligand for a transcription factor. For example, specifictoxic peptides may include peptide fragments from the Hanta virus orfrom the Bax molecule (abrogating Bcl-2 mediated cell survival); a toxiclipid ligand to a transcription factor might be a modulator of PPARactivity.

Very generally, intracellular routing sequences as contemplated herein,are compounds that affect the routing of a molecule within a cell. Theintracellular routing sequences are based on the seminal work of GuenterBlobel and colleagues in the field of intracellular protein topogenesis(Blobel, Proc. Natl. Acad. Sci. 77:1496-1500, 1980) who was awarded theNobel Prize for his discoveries. The inclusion of a routable structuralelement is contemplated to overcome technical limitations of earlierconcepts (e.g. Wu et al., J. Biol. Chem. 264:16985-16987, 1989, andrelated USP) where nucleic acids were rapidly degraded in lysosomes(discussion in Hart et al., Human Gene Therapy 9:575-585, 1998); Hart etal., Gene Therapy 2:552-554,1995), or e.g. Rodwell et al., whereimproved peptidomimetic targeting structures cannot be selected andcannot be expressed. It is appreciated in the art that intracellularrouting sequences are modular, i.e. they may be transferred to adifferent molecule while maintaining the operational characteristics oftargeting. The relatively short and physiological routing sequence(s)encompassed herein are contemplated to have less disruptive effects oncellular secretory pathways than viral (Wagner et al., Proc. Natl. Acad.Sci USA 89: 7934-38,1992) or bacterial (U.S. Pat. No. 5,643,599)sequences suggested previously. Such sequence 125 may be an oligopeptideor, preferably, a peptidomimetic connected to any bio-effective portionby any type of covalent bond. It is disclosed that intracellular routingsequences, being transferable to other protein/peptide environments, maybe used as a tool of targeted drug delivery. A variety of intracellularrouting sequences have been identified in different human proteins.Detailed analysis of the intracellular trafficking pathways has revealedexamples that typically, a combination of several short andnon-identical sequences act cooperatively. Such intracellular routingsequences include, but are not limited to: (a) cytoplasmic routingsequences, such as sequences targeting ribosomal/mRNA transportationcomplexes, and N-terminal or C-terminal cytoplasmic retention signals,b) ER-Golgi routing sequences, such as N- or C-terminal ER exportsignals, N- or C-terminal ER retention signals, N- or C-terminal ERretrieval sequences, N- or C-terminal targeting signals to theER/intermediate Golgi compartment (ERGIC), N- or C-terminal targetingsignals to cis-Golgi vesicles, N- or C-terminal targeting signals tomedial Golgi vesicles, N- or C-terminal targeting sequences totrans-Golgi vesicles or to the trans-Golgi network, network exportsignals, trans-Golgi network retrieval sequences, and (c) N- orC-terminal nuclear translocation sequences for nuclear import, nuclearexport, and nucleolar localization. Intracellular routing sequences maybe further enhanced by intramolecular sequences functioning asindependent elements or sub-elements that, e.g., break helical and betasheet structures and allow C-terminal ER retention signals to workespecially well (e.g. Lys-Pro).

Very generally, it is desirable to provide a DDM comprising: a BAM thatis coupled to a leader that does selectively bind to a somatostatinreceptor via a linkage enabler to a linker covalently connected with theleader; which linker comprises an intracellular routing or targetingsequence and a linkage enabler that is distinct from the BAM, from theintracellular targeting sequence and from the leader.

In certain preferred embodiments, the DDM is distinguished by comprisinganother independent structural element or sub-element that enables thenecessary performance and multifunctionality of the linker-BAMconnection, through the introduction of additional functional groups andresidues that are contiguous to intracellular targeting sequences, butfunctionally unrelated to intracellular routing. A structural elementwith such properties is henceforth termed linkage enabler. The functionof a linkage enabler is to control protection, activation andbioavailability of the BAM, and to provide attachment of a BAM to alinker at a stoichometric ratio of BAM:linker ≧1, under conditions ofoptimized BAM-linker geometry and controlled bioavailability of the BAM.The role of the linkage enabler for the BAM is similar to the role ofthe intramolecular sequence element for the intracellular targetingsequences. The linkage enabler does not specifically bind to any of theintracellular transport proteins, hence does not contribute tointracellular targeting of the presentation molecule, and isfunctionally distinct from intracellular targeting sequences,intramolecular sequence elements, and from a combination ofintracellular targeting sequences and intramolecular sequence elements.The linkage enabler may be see as an extension of the BAM, since itaffects the geometric position of the BAM in the presentation molecule,and controls the bioavailability of the BAM; the linkage enabler alsocooperates closely with the intramolecular sequence element which is asubelement of the linker, and may form an integrated structural andfunctional unit with the intramolecular sequence element.

Linkage enablers may consist of any number and kind of amino acids orresidues containing isosteric functional groups. In its simplest form, alinkage enabler comprises one or more bifunctional amino acids orsynthetic derivatives which are preferably connected to one or morebifunctional molecules (such as those termed “handles” in solid-phasechemistry, used for a wide array of covalent linkages) to achievelinkage to BAMs at a ratio of BAM:linkage enabler >1. All isomers,analogs and homologs of bifunctional amino acids are contemplated; forreasons of biological stability, non-amino acid derivatives arepreferred. Linkage enablers are discussed in detail in my copending U.S.patent application Ser. No. 10/910,218, the disclosure of which isincorporated herein by reference. One such linkage enabler isLys-Pro-Lys-Pro-Lys (SEQ ID NO: 35). Suitable intramolecular sequencesas described therein may also be used.

Routing to Golgi Subcompartments

Both C-terminal and N-terminal signals may be utilized for routing todifferent Golgi subcompartments. For example, the N-terminal 27 aminoacids of the glutamic acid decarboxylase isoform GAD65 function as amodule to target heterologous proteins primarily to regions of the Golgiapparatus that colocalize with Lens culinaris lectin binding sites(Michele et al., J. Cell Biol.126:331-341, 1994) predominantly found inthe cis-Golgi compartment and the adjacent distal medial Golgicompartment (Pavelka and Ellinger, J. Histochem. Cytochem. 37:877-894,1989). Thus, the GAD65 N-terminusMet-Ala-Ser-Pro-Gly-Ser-Gly-Phe-Trp-Ser-Phe-Gly-Ser-Glu-Asp-Gly-Ser-Gly-Asp-Pro-Glu-Asn-Pro-Gly-Thr-Ala-Arg(SEQ ID NO: 5), and functionally equivalent variations thereof, arecontemplated as a preferred routing signal for cis-Golgi and medialGolgi subcompartment.

Routing to the trans-Golgi network may be accomplished through aSer-X-Tyr-Gln-Arg-Leu (SEQ ID NO: 6) motif, such as theSer-Asp-Tyr-Gln-Arg-Leu (SEQ ID NO: 7) motif in protein TGN38 (see e.g.Wang et al., J Biol. Chem. 268:22853-22862, 1993) which routes theprotein back into the TGN through the early endosome compartment, orthrough the acidic cluster motif Cys-Pro-Ser-Asp-Ser-Glu-Glu-Asp-Glu-Gly(SEQ ID NO: 8) (residue 774 to 783 in the C-terminus of the endoproteasefurin; Schaefer et al., EMBO J. 14:2424-2435,1995). Phosphorylation ofthe acidic cluster motif of furin (see SEQ ID NO: 24) by casein kinaseII facilitates retrieval from clathrin-coated immature vesicles into theTGN by assembly protein 1. The acidic cluster motif cooperates with asecond upstream sequence of the type Leu-Ile-X-Tyr-Lys-Gly-Leu (SEQ IDNO: 9) (residue 759 to 765 of furin) which comprises an imperfectleucine motif and an endocytosis motif Tyr-Lys-Gly-Leu and also binds tothe assembly protein 1 complex. Targeting to the TGN may be enhanced bycombining a retrieval motif such as Ser-Asp-Tyr-Gln-Arg-Leu (SEQ ID NO:7) with a membrane-spanning sequence, such asPhe-Phe-Ala-Tyr-Leu-Val-Thr-Ala-Ala-Val-Leu-Val-Ala-Val-Leu-Tyr-Ile-Ala-Tyrfrom TGN38 (SEQ ID NO: 10).

Routing to different subcompartments of the Golgi apparatus may furtherbe accomplished by using signals encoded in the transmembrane domainsand certain adjacent sequences of resident Golgi proteins, such asenzymes participating in posttranslational modification byglycosylation. It is known that processing of carbohydrate side chainscorrelates with a distinct topology within the ER and the Golgiappparatus, such that UDG-glucuronosyltransferase can be used as amarker for ER, N-acetylgalactosamine transferase activity as a markerfor late pre-Golgi/early Golgi compartments,N-acetylglucosaminyltransferase I activity as marker for the medialGolgi compartment, beta-1,4-galactosyltransferase as marker fortrans-Golgi cisternae, and beta-galactoside-alpha-2,6-sialyltransferaseas marker for trans-Golgi network (TGN). It has been shown that theN-terminal 33 amino acids, or the signal anchor domain, ofbeta-galactoside-alpha-2,6-sialyltransferase (for the human enzyme:Met-Ile-His-Thr-Asn-Leu-Lys-Lys-Lys-Phe-Ser-Cys-Cys-Val-Leu-Val-Phe-Leu-Leu-Phe-Ala-Val-Ile-Cys-Val-Trp-Glu-Lys-Lys-Gly-Ser-Tyr-Tyr-Asp;SEQ ID NO: 11), comprising the cytoplasmic domain and transmembranedomain with six C-terminally adjacent amino acids, can be transferred toreplace the corresponding domains of the cell surface enzyme dipeptidylpeptidase IV, and will convert the resulting chimeric enzyme to a TGNresident protein. If the cytoplasmic sequence context of dipeptidyltransferase IV is maintained, the necessary transmembrane domain frombeta-galactoside-alpha-2,6-sialyltransferase can be reduced to a routingsequence of 17 amino acids that functions as a TGN retention signal.Transmembrane domains of Golgi glycosylation-modifying enzymes aretypically highly conserved among species and are distinct for eachenzyme (see Wong et al., J. Cell Biol. 117:245-258, 1992). It iscontemplated that signal anchor domains, in particular transmembranedomains with adjacent amino acids, of all resident Golgiglycosylation-modifying enzymes comprise suitable routing sequences fordistinct subcompartments of the Golgi apparatus, often with narrowersubcompartment selectivity than viral proteins defining viral buddingregions within the Golgi apparatus. It is further contemplated that thedrug delivery molecules of the invention may comprise an intracellularrouting sequence derived from a transmembrane domain of aglycosylation-modifying resident Golgi enzyme.

Targeting to different Golgi subcompartments may also be achieved byusing signals derived from pathogenic viruses. It is known thatcoronaviruses, cytomegalovirus, poliovirus, and vaccinia virus bud fromthe ER-intermediate Golgi compartment, whereas rubella virus buds fromGolgi compartments. The budding site of a virus may not always bepredictive for the routing of individual proteins; for example, therouting of rubella virus protein E1 (tubular ER exit sites/ERGIC; Hobmanet al., Mol. Biol. Cell 9:1265-78, 1998) and the final assembly/buddingof the complete virus (Golgi compartment) differ in location. Thecomplex formation of several viral proteins, each having differingtargeting motifs, within the budding virus may also restrict therepertoire of functional targeting motifs, leading to a preferredbudding site more or less narrowly defined within the Golgi compartment.For example, members of the Bunyavirus family, including the Uukuniemivirus, typically bud from the Golgi, however, a very broad Golgi routingselectivity has been observed for a 81-amino acid domain from thecytoplasmic tail of the Uukuniemi G1 glycoprotein, with preference forthe mid-Golgi compartment, but significant targeting to ERGIC and TGN aswell (Andersson and Pettersson, J. Virol. 72:9585-9596, 1998). Theminimum sequence with full Golgi routing capacity was defined asThr-Trp-Lys-Ile-Ile-Lys-Pro-Phe-Trp-Trp-Ile-Leu-Ser-Leu-Leu-Cys-Arg-Thr-Cys-Ser-Lys-Arg-Leu-Asn-Arg-Ala-Glu-Arg-Leu-Lys(SEQ ID NO: 12). C-terminalLys-Tyr-Lys-Ser-Arg-Arg-Ser-Phe-Ile-Asp-Glu-Lys-Lys-Met-Pro (SEQ ID NO:13) is an intermediate Golgi targeting/retention signal (Lotti et al.,J. Biol. Chem. 274:10413-10420, 1999). A partially homologous sequencecan also be found in rubella virus E1 protein, as part of a largersequenceTrp-Trp-Asn-Leu-Thr-Leu-Gly-Ala-Ile-Cys-Ala-Leu-Pro-Leu-Val-Gly-Leu-Leu-Ala-Cys-Cys-Ala-Lys-Cys-Leu-Tyr-Tyr-Leu-Arg-Gly-Ala-Ile-Ala-Pro-Arg(SEQ ID NO: 14) which was identified to afford ER retention (Hobman etal., J. Virol. 71:7670-7680, 1997). All viral protein sequencescompetent to afford routing to specific Golgi subcompartments arecontemplated.

Routing to Late Golgi, Post-Golgi, and Specialized Vesicles

It is thought that vesicular targeting proteins (VAMPs, SNAPs, v-SNAREs)in TGN-bound vesicles, or also in post-Golgi vesicles, bind to adaptorproteins to generate the necessary routing specificity for a givenvesicle, such that reaching the appropriate destination of the vesicleis ascertained on the molecular level by recognition of a cognateresident docking receptor (syntaxin or t-SNARE). It is furthercontemplated that vesicular targeting, or targeting of cargo typicallytransported in a certain type of vesicle, may be accomplished bypeptides or peptidomimetic structures interacting with resident syntaxinmolecules. As an example, ER-to-TGN targeting involves the highlyconserved H3 domain of syntaxin 5 with its coiled-coil motif that issufficient for binding of vesicles. Peptidomimetic structures bindingthe sequenceThr-Arg-His-Ser-Glu-Ile-Ile-Lys-Leu-Glu-Asn-Ser-Ile-Arg-Glu-Leu-His-Asp-Met-Phe-Met-Asp-Met-Ala-Met(SEQ ID NO: 15) of syntaxin 1a (Kee et al., Neuron 14:991-998, 1995) area highly preferred embodiment for docking a drug delivery molecule tothe TGN compartment. It is contemplated that structures resembling thesequence 1-27 of GAD65 (SEQ ID NO: 5) may encode certain syntaxindocking capabilities, including binding to syntaxin moleculessurrounding synaptic vesicles.

Lysosomal targeting from the TGN through the late endosomal compartmentmay be accomplished by the sequenceArg-Lys-Arg-Ser-His-Ala-Gly-Tyr-Gln-Thr-Ile (SEQ ID NO: 16) (lamp-1;Höning et al., EMBO J. 15:5230-5239, 1996 and references therein). It isspecifically contemplated to target proteins involved in pathologicalpathways, in particular pathways of cell proliferation and cellinvasiveness, for accelerated degradation in lysosomal vesicles orproteasomes. Preferred embodiments are drug delivery molecules carryingthe lysosomal targeting sequenceArg-Lys-Arg-Ser-His-Ala-Gly-Tyr-Gln-Thr-Ile (SEQ ID NO: 16) or afunctional equivalent fused to a peptide or peptidomimetic with highbinding affinity to any of the cancer-associated orangiogenesis-associated growth factor receptors or signaling molecules.Alternatively, drug delivery molecules may carry poly-ubiquinatedresidues fused to a high-affinity binding moiety interacting with agrowth factor or signaling molecule. Any molecule havingpathophysiological significance in any disease context is contemplated.

Routing to Polarized Cellular Regions

The TGN sorting function (reviewed in Keller and Simons, J. Cell Sci.110:3001-3009, 1997) is particularly important, because it contributesto the maintenance of plasma membrane asymmetry in polarized cells whichhave a barrier function and regulate macromolecular flow, such asvectorial transport, as well as compartment-specific signaling betweendifferent organ compartments. Significantly perturbed membrane polarityis a condition of cellular pathology. N-terminal basolateral sortingsignals have been identified in transferrin receptor and the invariantchain of the major histocompatibility antigen class II, whereasC-terminal basolateral sorting signals have been identified in LDLreceptor and lysosomal acidic phosphatase. Typically, multiple parallelsorting pathways exist. Transcytosis of a presentation molecule throughendothelia may be accomplished by using one or more basolateraltargeting sequences. This may be particularly useful for delivery ofdiverse BEMs to cell types distal to an endothelial cell overexpressingsomatostatin receptors (or other surface molecules amenable to selectivetargeting), such as neurons under survival stress in Alzheimer'sdisease.

Contemplated Embodiments

Contemplated for diagnostic and therapeutic purposes are all forms ofnucleolar, nuclear, and extranuclear mRNA targeting. Specificallycontemplated is the use of peptidomimetic or peptidic targetingsequences mimicking any targeting structures afforded by proteincomplexes to target mRNA to ribosomes, including structures mimickingthose afforded by Pur-alpha, staufen, and mRNA targeting complexescomprising Pur-alpha and/or staufen. Particularly preferred aretargeting peptides comprising short repeats of the type Met-X-Arg orMet-X-Lys and the sequence Leu-Met-Tyr-Arg-Leu-Tyr-Met-Ala-Glu-Asp (SEQID NO: 17) for direct ER targeting in the context of any therapeuticintervention at a co-translational or post-translational stage, such asmodulation of nascent peptide chains, co-translational delivery to theER (or ER-ribosome complex) of a wild-type peptide fragment spanning amutation (e.g. a mutation causing misfolding of a transmembranecoregulator protein in cystic fibrosis) whereby the wild-type fragmentmay participate in post-translational repair processes in the ER, andthe like. The sequences Met-X-Arg, Met-X-Lys, andLeu-Met-Tyr-Arg-Leu-Tyr-Met-Ala-Glu-Asp (SEQ ID NO: 17) are furtherpreferred for delivering to the ER modulators of the prolyl-cis-transisomerization pathway, or of the ubiquitination pathway targetingproteins to the proteasome degradation pathway. Peptidomimeticstructures of staufen and Pur-alpha are preferred overLeu-Met-Tyr-Arg-Leu-Tyr-Met-Ala-Glu-Asp (SEQ ID NO: 17) to deliver sensenucleic acid sequences to active ribosomes for repair of geneticdiseases or expression of anti-apoptotic genes, or for delivery ofantisense nucleic acid sequences, ribozymes, or aptamers toER-associated ribosomes to inhibit expression of disease-associatedgenes of pathogenetic significance, such as growth factors and growthfactor receptors, kinases and signaling molecules in cancer andpathological angiogenesis, or viral genes following infection with ahepatitis virus or an HIV virus. Preferred embodiments of drug deliverymolecules are those that achieve ribozyme targeting to nucleus andcytoplasm; antisense nucleic acid derivative targeting to nucleus,cytoplasm and particulate ribosomes; targeting of small interfering RNAto cytoplasm.

Contemplated further is targeting of all compartments of ER and Golgiwith appropriate peptidic signals and peptidomimetic derivativesthereof, e.g. to prevent or ameliorate harmful misrouting of metabolitesin ER storage diseases; to interfere with sorting of oncogenes,constitutively activated kinases, growth factors and growth factorreceptors in cancer; to deliver carbohydrate building blocks (lipidbuilding blocks) in diseases linked to aberrant glycosylation (lipidmetabolism); to deliver small molecules to compensate for misfoldedprotein species. Specifically contemplated are drug delivery moleculesbearing a TGN targeting sequence linked to a branched mannosyl buildingblock; drug delivery molecules comprising stable peptidomimetic growthfactor receptor ligands as a BAM, connected to a linker bearing a TGNexit signal and a lysosomal targeting sequence, to misrouterecirculating growth factor receptors in cancer cells towards adegradation pathway; drug delivery molecules comprising inhibitorysubstrates for PI3K or Akt as a BAM, connected to a linker bearing alysosomal targeting sequence. The combination of TGN exit andcytoplasmic retention signals is contemplated to be particularly useful.The combination of ER exit signal and cytoplasmic retention signaldefines sequential targeting action; it is contemplated that suchtargeting action may be functionally further improved by addition of afurther signal for secretion (TGN exit) in order to perform best.

Contemplated further is routing to all compartments and pathways outsideof ER and Golgi apparatus, including, but not limited to, endosomes(contemplated routing method is, e.g., by ligands to endosome-specificmarkers from the rab family); cell-specific synaptic and post-synapticvesicles (contemplated method by syntaxin ligands); regions of polarityin cells (contemplated method by e.g. basolateral sorting signals); cellsurface (e.g. contemplated method by modification of signalsparticipating in endocytic transport and ER exit).

Examples of C-terminal ER-intermediate Golgi Compartment Routing Signals(SEQ ID NO: 18) Lys-Pro-Lys-Cys-Pro-Glu-Leu-Pro-Pro-Phe-Pro-Ser-Cys-Leu-Ser-Thr-Val-His-Phe-Ile-Ile-Phe-Val-Val-Val-Gln-Thr-Val-Leu-Phe-Ile-Gly-Tyr-Ile-Met-Tyr-Arg-Ser-Gln-Gln-Glu-Ala-Ala-Ala-Lys-Lys-Phe-Phe- Ala-Ala-Ala

-   Cys-Lys-Tyr-Lys-Ser-Arg-Arg-Ser-Phe-Ile-Asp-Glu-Lys-Lys-Met-Pro (SEQ    ID NO: 19) (Adenovirus E19 protein; Nilsson et al., Cell 58:707-718,    1989)-   Lys-Tyr-Lys-Ser-Arg-Arg-Ser-Phe-Ile-Asp-Glu-Lys-Lys-Met-Pro (SEQ ID    NO: 13) (Adenovirus E19 protein; Lotti et al., J. Biol. Chem.    274:10413-10420, 1999)    Ile-Tyr-Ile-Trp-Ala-Pro-Leu-Ala-Gly-Thr-Cys-Gly-Val-Leu-Leu-Leu-Ser-Leu-Val-Ile-Thr-Lys-Tyr-Lys-Ser-Arg-Arg-Ser-Phe-Ile-Asp-Glu-Lys-Lys-Met-Pro    (SEQ ID NO: 20) fusion peptide CD8alpha-transmembrane domain/ad19    (Lotti et al., J. Biol. Chem. 274:10413-10420, 1999)-   Lys-Tyr-Lys-Ser-Arg-Leu-Gln-Gly-Ala-Cys-Thr-Lys-Lys-Thr-Ala (SEQ ID    NO: 21) HMGCoA-reductase (Jackson et al., J. Cell Biol. 121:317-333,    1993)-   Trp-Trp-Asn-Leu-Thr-Leu-Gly-Ala-Ile-Cys-Ala-Leu-Pro-Leu-Val-Gly-Leu-Leu-Ala-Cys-Cys-Ala-Lys-Cys-Leu-Tyr-Tyr-Leu-Arg-Gly-Ala-Ile-Ala-Pro-Arg    (SEQ ID NO: 14) rubella virus E1 protein (Hobman et al., J. Virol.    71:7670-7680, 1997)    Example of N-terminal Cis-Golgi/Medial Golgi Compartment Routing    Signal-   Met-Ala-Ser-Pro-Gly-Ser-Gly-Phe-Trp-Ser-Phe-Gly-Ser-Glu-Asp-Gly-Ser-Gly-Asp-Pro-Glu-Asn-Pro-Gly-Thr-Ala-Arg    (SEQ ID NO: 5) (residues 1-27 of GAD65; Michele et al., J. Cell    Biol.126:331-341, 1994)

Examples of C-terminal Cis-Golgi Compartment Routing Signals (SEQ ID NO:22) Gln-Asn-Gly-Ser-Lys-Pro-Lys-Cys-Pro-Glu-Leu-Pro-Pro-Phe-Pro-Ser-Cys-Leu-Ser-Thr-Val-His-Phe-Ile-Ile-Phe-Val-Val-Val-Gln-Thr-Val-Leu-Phe-Ile-Gly-Tyr-Ile-Met-Tyr-Arg-Ser-Gln-Gln-Glu-Ala-Ala-Ala (SEQ ID NO: 23)Lys-Pro-Lys-Cys-Pro-Glu-Leu-Pro-Pro-Phe-Pro-Ser-Cys-Leu-Ser-Thr-Val-His-Phe-Ile-Ile-Phe-Val-Val-Val-Gln-Thr-Val-Leu-Phe-Ile-Gly-Tyr-Ile-Met-Tyr-Arg-Ser-Gln-Gln-Glu-Ala-Ala-Ala-Lys-Ser-Phe-TyrExample of C-terminal Medial Golgi Compartment Routing Signal

-   Thr-Trp-Lys-Ile-Ile-Lys-Pro-Phe-Trp-Trp-Ile-Leu-Ser-Leu-Leu-Cys-Arg-Thr-Cys-Ser-Lys-Arg-Leu-Asn-Arg-Ala-Glu-Arg-Leu-Lys    (SEQ ID NO: 12) (Uukuniemi virus glycoprotein G1, cytoplasmic tail;    Andersson and Pettersson, J. Virol. 72:9585-9596, 1998)    Examples of C-terminal Trans-Golgi Network Routing Signals-   Ser-X-Tyr-Gln-Arg-Leu (SEQ ID NO: 6)-   Ser-Asp-Tyr-Gln-Arg-Leu (SEQ ID NO: 7) (human TNG protein 2/TGN38;    Wang et al., J Biol. Chem. 268:22853-22862, 1993)-   Cys-Pro-Ser-Asp-Ser-Glu-Glu-Asp-Glu-Gly (SEQ ID NO: 8) (residue 774    to 783 in the C-terminus of the endoprotease furin; Schaefer et al.,    EMBO J. 14:2424-2435, 1995).-   Cys-Pro-(Phospho-Ser)-Asp-(Phospho-Ser)-Glu-Glu-Asp-Glu-Gly (SEQ ID    NO: 24) (phosphorylated residue 774 to 783 C-terminal furin signal)    Example of Trans-Golgi Exit/Lysosomal Routing Signals

Arg-Lys-Arg-Ser-His-Ala-Gly-Tyr-Gln-Thr-Ile (SEQ ID NO: 16) (lamp-1;Höning et al., EMBO J. 15:5230-5239, 1996)

Examples of Nucleolar/Nascent Ribosome Routing Signals:

N-terminal

-   Lys-Lys-Lys-His-Ser-His-Arg-Gln-Asn-Lys-Lys-Lys-Gln-Leu-Arg-Lys (SEQ    ID NO: 25) (residue 24-39 of DDX10 RNA helicase)    C-terminal-   Lys-Lys-Lys-Met-Thr-Lys-Val-Ala-Glu-Ala-Lys-Lys-Val-Met-Lys-Arg (SEQ    ID NO: 26) (residue 665-680 of DDX10 RNA helicase)    Example of ER-Associated Ribosome Routing Signals-   Met-X-Arg (SEQ ID NO: 27); Met-X-Lys (SEQ ID NO: 28) and    Leu-Met-Tyr-Arg-Leu-Tyr-Met-Ala-Glu-Asp (SEQ ID NO: 17) (human    cytochrome b₅ (125-134); Mitoma and Ito, EMBO J 11:4197-4203, 1992)    Examples of Nuclear Translocation Signals-   Arg-Arg-Ser-Met-Lys-Arg-Lys (SEQ ID NO: 29) (Hsieh et al., J. Cell.    Biochem. 70: 94-109, 1998)-   Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO:30) and related bipartite    consensus sequences as described in Dingwall and Laskey, Trends    Biochem Sci. 16:478-481, 1991; Yamanaka et al., J. Biol. Chem. 269:    21725-21734, 1994 (hereby incorporated by reference)-   KKRKLIEENPKKKRKV (SEQ ID NO: 4)    and other sequences described in Jans et al., Medicinal Research    Reviews 18:189-223, 1998 (hereby incorporated by reference).    Guided Cell Destruction by BNCT

The drug delivery molecule is particularly designed for guided celldestruction. U.S. Pat. No. 6,074,625 indicates that certainboron-containing compounds can be targeted to hormonally responsivecells containing steroid hormone receptors which are predominantlynuclear, by contacting those cells with a boron-containing agent whichis conjugated to a ligand having binding specificity for the particularintercellular receptor of the cell, and it advocates using syntheticsteroid analogs as ligands that are preferably anti-receptor agents orantagonists. It is also indicated that, alternatively, boron compoundscan be employed for diagnosis of properties, and their locations can bedetermined by magnetic resonance imaging (MRI). The shortcomings of thetechnology presented in U.S. Pat. No. 6,074,625 are that steroidreceptors are rather ubiquitous, that selective ligands have to entertarget cells by diffusion, and that specific cytoplasmic bindingproteins for a number of steroid hormones have been identified, thusgenerating a very high and long-lasting background level of boron innon-target cells. Furthermore, the additional functionality of a routingmoiety to achieve improved targeting to subcellular topographies isabsent in U.S. Pat. No. 6,074,625. In contrast, the current inventionteaches the use of peptidic or peptidomimetic non-agonist ligandscognate to surface receptors with a restricted expression profile, andpresents the advantage of target cell selectivity at the level ofradiopharmaceutical uptake.

U.S. Pat. No. 5,846,741 discusses the rather cumbersome concept oftissue pre-targeting by an adaptor molecule for in vitro imagingapplications prior to using BNCT to be certain that sufficient boron-10atoms are delivered to the tumor site. The pre-targeting is carried outby administering a tagged antibody that will bind to antigens producedby or associated with the tumor cell. In a second step, an compositionis administered wherein a boron-containing compound is conjugated to amolecule with high affinity for the tag existing on the previously boundantibody, causing the boron compound to be localized at the sites of thepre-treatment. As an example, the use of a known tagging pair, such asbiotin and avidin, is suggested. It is also suggested that a doublelabeled biotin can be used, e.g. one that would carry both boron andI¹²⁵. The use of I¹²⁵ would allow one to determine that the boroncompound had reached the desired tumor; moreover, monitoring could tellhow long it would take for the label to be cleared from non-targetorgans. Disadvantages of the procedure are that the systemic use ofantibodies is expensive and fraught with the risk of forming antibodies.The efficacy and selectivity of boron targeting as exemplified in U.S.Pat. No. 5,846,741 is conceptually problematic. Avidin occurs naturallyin serum and will compete with a biotinylated boron composition, therebyreducing the amount of boron composition available for binding to anavidin-antibody conjugate. It is well known that cells of many abundanttissue types, e.g. skeletal muscle and intestines, contain a sizablepool of biotin. The boron-modified biotin may be taken up into that poolin the absence of a tumor-specific antibody, turning e.g. skeletalmuscle (through which the neutron beam invariably has to pass on its wayto the tumor) into a target for destruction. Moreover, if a biotinylatedantibody is used, the boron-conjugated avidin composition is a target ofproteolytic degradation, and the avidin may bind to cellular biotin onthe surface of or within non-tumor tissues, again causing seriousmistargeting. Since only antibodies to recirculating membrane epitopesmay enter the cell, under which condition the binding tags are no longeraccessible to the affinity partner in the second stage of the treatmentcomposition, virtually all antibody-bound boron compounds will nevergain entry into the target cell. Instead, they will mostly causedestruction in the extracellular space, since only one out of sixpossible flight directions of the alpha particle may hit the targetcell, while the energy of most alpha particles is quickly absorbed byproteins and liquid in the extracellular space. Thus, it has been feltnecessary to improve on the technology described in U.S. Pat. No.5,846,741. A critical simplification enhancing targeting efficiency isfelt to be the use of a single compound without antibody characteristicswhich entails cellular entry into predominantly tumor cells and tumorvasculature.

An article by C.S. Zuo entitled “Protron nuclear magnetic resonancemeasurement of p-boronophenylalanine (BPA): A therapeutic agent forboron neutron capture therapy”, Med. Phys. 26 (7) 1230-1236, describes aneed to be able to determine that the desired concentration of the B10that has accumulated at the tumor. Generally the boron that is employedfor BNCT is greater than 98% isotopically enriched in the B10 isotopewhich undergoes the desired fission reaction upon thermal neutroncapture. Because it was so difficult to actually determine the boronconcentration by NMR, instead it is suggested that proton NMR be used todetect the protons associated with the BPA carrier molecule. Althoughthis attack is perhaps feasible, it is executed only with somedifficulty. As an alternative, monitoring via neutron captureradiography (NCR) to determine when boron was carried to a tumor usingBPA was advocated as a better non-invasive way of determining theconcentration of boron that had reached the tumor; however, this againis not considered to be easily executed.

In alternative therapies, investigations were carried out with respectto the use of a number of radionuclides, and the possibility of usingsome of these nuclides for therapy and simultaneous PET diagnosis wassuggested, see Stephanek et al. “Auger-Electron Spectra of Radionuclidesfor Therapy and Diagnostics” Acta Oncologica, 35 (7) 863-868, (1996).Two articles in 1999 by H. Lundqvist and M. Lubberink et al. discussPET, see Acta Oncologica, 30 (3) pp. 335-341 (1999). The articlesindicate that radionuclides for internal therapy usually do not emitpositrons, while PET is a good tool that can be employed to obtain anaccurate determination of a regional absorbed dose reaching a targetedorgan of a patient. The paper suggests performing a PET investigation inadvance to allow dose-planning under identical conditions that will beused in the therapy. It indicates that ¹²⁴I has an ideal half life thatcould be used to measure the full radioactivity integral of a commonlyused therapeutic nuclide such as ¹³¹I. It is also suggested that a smallamount of ¹²⁴I might be included along with ¹³¹I to make it possible toconduct a PET study during therapy.

Whereas previously it has been necessary to employ such a dose-planningregimen where two sequential administrations are made to a patient (thisapproach has been used therapeutically in U.S. Pat. No. 5,846,741),applicant has now found that it is feasible to employ a combination ofPET and BNCT by using a unique drug delivery molecule (DDM) wherein boththe accurately detectable label and the cell-destructive molecule aresimultaneously targeted to a organ or tissue. By linking the DDM to areceptor (without activating that receptor) via a selective targetingmoiety, causing receptor-dependent internalization, and further causingthe DDM to accumulate within a pre-determined subcellular compartmentvia a routing moiety, this objective can be efficiently accomplished.Then, by using PET or SPECT, it can be accurately determined whether adesired concentration has been achieved. Thereafter, assuming BNCT isbeing employed, a beam of thermal neutrons can be focused onto theparticular organ so the boron compounds which accumulated are actuated;upon fission of the boron nuclei, cell-destructive alpha particles arecreated, leading to destruction of single target cells, but not ofadjacent biological structures, the integrity of which may be criticalto a patient's survival (e.g. major blood vessels, ureter, alveoli andacini in the lung, etc.). The benefit of this particular combination ofmodalities provided by applicant's invention lies in conditionallyharnessing the destructive power of an alpha particle following acontrolled targeting event that is monitored and validated at highresolution by a functional imaging modality. This is in contrast to theimaging modality of conventional CT, that provides information aboutanatomical structures without identifying those structures functionallyand without having the resolution power of targeted PET (SPECT) imagingof single cell or subcellular structures, as practiced in thisinvention, which facilitates critical validation by identifying thedesired target environment by way of its subcellular localization, andoptionally by way of its functional properties.

A variety of boron compounds are well known for use in BNCT. They can bedivided into mainly four groups: boronated nucleosides, boronatedporphyrins, boronated amino acids, and sodium borocaptate (Lu et al.,Adv. Drug Deliv. Rev. 26, 231-247, 1997). Examples for boronatednucleosides are beta-5-o-carboranyl-2′-deoxyuridine (Hurwitz et al.,Nucleosides Nucleotides Nucleic Acids 19, 691-702, 2000) and N-3substituted carboranyl thymidine analogs (Tjarks et al., NucleosidesNucleotides Nucleic Acids 20, 695-698, 2001). Examples for boronatedporphyrins are tetraphenyl-carborane-porphyrines and boronatedprotoporphyrin (BOPP). BOPP has been evaluated as a potential drug fordual BCNT and photodynamic therapy (Callahan et al, Int. J. Radiat.Oncol. Biol. Phys. 45, 761-771, 1999) and was found to accumulate inlysosomes, but not in mitochondria or the nucleus. Cellular uptake ofBOPP was dependent on LDL receptor which is not a tumor-selectivereceptor. Consequently, the cellular selectivity and intracellulardistribution of free BOPP is very unfavourable. From a synthetic anddrug delivery perspective, relatively large complexes with multipleboron substitutions afford the most obvious way to introduce a maximumof boron atoms into a tumor cell. However, such complexes are typicallydifficult to synthesize and display considerable toxicity. Recently,bisphosphonate derivatives of dodecahydro-closo-dodecaborate have beendemonstrated to have acceptable toxicity (Tjarks et al., Anticancer Res.21, 841-846, 2001). Liposomal enclosure of o-carboranylpropylamine ledto a loading density of 13,000 molecules per vesicle at acceptabletoxicity, suggesting that conjugation of that compound instead ofenclosure may also reduce toxicity. Borylated ferrocenium compounds arecomparatively facile to synthesize and offer a high number ofsubstituted boron atoms per molecule; however, a first series of twelvederivatives did not appreciably accumulate in tumors (Weissfloch et al.,Biometals 14, 43-49, 2001). Boronated DNA-intercalating compounds, suchas 5-o-carboranyl phenanthridium and 6-p-carboranyl-phenanthridium,showed limited uptake into cultured human glioma cells; low toxicitycorrelated with low boron accumulation (Gedda et al., Anticancer DrugDes 15, 277-286, 2000).

It is specifically contemplated that all these compounds can becovalently linked as a part of the drug delivery molecule (DDM), andapplied to BNCT. The labels and a boron-containing moiety (when such isbeing used) may be attached independently, and preferably covalently, asa part of the drug delivery molecule at any location (i.e., at thetargeting, routing, or BAM moiety). PET imaging using contemplated drugdelivery molecule is desired, and the PET label is preferably attachedto the routing moiety. Particularly preferred labels includetyrosine-bound I¹²³ and/or I¹²⁴; some of the tyrosines may be a part ofor attached to the routing moiety. Where slow neutron capture is to beused to effect subsequent cell destruction, all known natural boronisotopes, as part of suitable compounds, are considered suitable foruse. Such compounds may be attached as part of the drug deliverymolecule by methods well known in the art. For example,borono-phenylalanine (BPA) may be linked to a routing molecule that isin turn covalently linked to a receptor-selective ligand, such as oneselective for SSR-2.

While BPA is one the best characterized compounds for BNCT, it hascertain disadvantages. BPA has been tested in tumor models andindividual patients and found to have a tumor enrichment factor of about3 or less, meaning that a substantial amount of boron is delivered tonon-target tissues. Clinical success of BNCT seems to depend on a tumorenrichment factor of the boron compound exceeding 3 (Barth et al, Cancer70, 2995-3007, 1992). Nguyen et al (Biochem Pharmacol. 45, 147-155,1993) have demonstrated that BPA uptake into whole glioblastoma cells invitro reaches its maximum after 1 h, with a cytosolic concentration of250 ng per million of glioma cells, while concentrations in nuclei,lysosomes and microsomes were 80-fold lower, 100-fold lower, and125-fold lower, respectively.

Improving the performance of radiopharmaceuticals for BCNT is animportant need to be met. The variability in tumor enrichment of BPAobserved in clinical patients by Kabalka et al (J. Nucl. Med. 38,1762-1767, 1997) underscores the need to identify reagents with a higherand more consistent tumor enrichment factor. A different type of BNCTreagent, beta-5-o-carboranyl-2′-deoxyuridine (D-CDU) has been found tohave a tumor enrichment factor in the range of 100, but showed increasedaccumulation in nontumor brain tissue at concentrations of 150 mg/kg(Schinazi et al, Clin. Cancer Res. 6, 725-730, 2000). The ¹⁰B enrichmentof 20% used was too low to reach therapeutically satisfactory boronconcentrations and led to no significant benefit over neutron treatmentalone. It remains to be determined whether higher enrichment with ¹⁰Bwill allow to optimize the dosage such that a sufficient intratumoral¹⁰B level is maintained when less than 150 mg/kg are administered.

The relatively low tumor enrichment factor of BPA, plus the cumulativetoxicity of boron compounds (Aziz et al, J. Neuropathology Exp. Neurol59, 62-73, 2000), limits the option to improve tumor saturation byincreasing the total amount of BPA administered. The broad tissuedistribution of BPA-like boron compounds bears the risk of substantialtissue damage upon exposure to epithermal neutrons. Planning andperformance of a suitable neutron capture treatment using fluorine-18(¹⁸F)-labeled BPA-like compounds thus requires optimization of a complexpharmacokinetic scenario, as described by Imahori et al (1998b), wherebythe neutron radiation has to be applied during a narrow time window whenwashout of BPA in non-target tissue has occurred, while concurrentmetabolism, decay of the short-lived ¹⁸F isotope, and leakage of BPAfrom target cells still provides for an acceptable signal-to-noiseratio. Imahori et al found further that there is no similarly fastdegradation, or reversal of uptake, in non-target tissues as is found intumor cells, the level of BPA in the tumor declines within several hoursto the level of BPA in healthy surrounding tissue that appears toincrease. Tissue destruction and tissue repair processes, likedevelopment of inflammatory response and removal of cellular debris bymacrophages, take several hours to become fully established, thusrendering unfeasible the real-time monitoring of BNCT efficacy by meansof measuring the concentration of doubly-labeled ¹⁸F-¹⁰B free amino acidin the tumor (Imahori 1998b).

An added complication is the strict dependency of BPA enrichment onamino acid transport systems in the tumor cell plasma membrane, asobserved by Imahori et al (1998b), which makes staging and grading, aswell as estimates of the tumor size, prone to large errors. Thus, PETbased on coupling of PET-competent radionuclids with a short half lifeto free amino acids is not a reliable strategy.

The current invention improves on the procedure described by Imahori etal, by pursuing an entirely different mechanistic approach to accumulatedoubly-labeled PET/BNCT drugs in tumor cells, namely byreceptor-mediated uptake and subsequent routing to subcellularcompartments. Furthermore, it entails utilization of SPECT imaging whichhas not previously been suggested for monitoring BNCT, or alternativelyPET-competent radionuclides with longer half-life, such as ⁶⁴Cu, whichwill allow to observe the elimination of tumor cells through a secondmodality in real-time. A decisive difference in the invention, whencompared to earlier disclosures, is its suitability for anti-angiogenicapplication of BNCT. The uptake of BPA, and numerous other BNCT drugscurrently under development, depends partially on the altered membranepotential of tumor cells, and also on over expression of amino acidtransporters understood as a property of tumor cells which isresponsible for the observed selectivity in BNCT drug uptake.

Amino acid transporter systems are highly diverse families of membraneproteins with overlapping specificities. Three major groups have beenidentified: A, L, ASC (Wagner et al, Am J Physiol Cell Physiol 281,1077-1093, 2001). It is a general principle that the transporters form aheterodimeric or -oligomeric complex with a heavy-chain molecule locatedin the plasma membrane; it is the complex that defines functionality(Pfeiffer et al, EMBO J 18, 49-57, 1999); much of the activityregulation in amino acid transporters appears to occur occurspost-transcriptionally (see also Freeman and Mailliard, Biochem BiophysRes Comm 278, 729-732, 2000 for further examples). Thus, L transportermRNA expression by itself is not sufficiently predictive for the levelof active L transporter protein in a given cell type. Systematic studiesof BPA uptake and molecular types of transport proteins responsible arerestricted to in-vitro studies. The closest structural analogs of BPAhave been found to be transported by L system and ASC system (Samnick etal, Nucl Med Biol 28, 13-23, 2001), whereas BPA has been found to betransported by L system and A system (Wittig et al, Radiat Res 153,173-180, 2000). While mRNA species for two molecular subtypes of the Ltransporter have been described in endothelia of the BBB, there iscurrently no agreement which subtype of L transporter is functionallydominant in BPA transport across brain endothelia of the BBB.Conflicting reports have emphasized predominance of subtype 2 (Segawa etal., J Biol. Chem. 274 19745-19751, 1999) or subtype 1 (Boado et al,Proc Natl Acad Sci USA 96, 12079-12084, 1999) in performing largeneutral amino acid transport in endothelia. However, consistent with theknown post-transcriptional regulation of transporter activity,functional evidence for over expression of L transporter protein in vivohas neither been found in BBB endothelia nor in tumor endothelia, basedon several methodologically different studies on rat models and patienttissues. Yang et al, Neurosurgery 47, 189-197, 2000 describe the need tochallenge the BBB with a bradykinin agonist in order to improve BPAtransport into the CSF and the glioblastoma. If the L system inendothelia surrounding the tumor was substantially more activated thanin nontumor tissue, or even activated to a similar degree as inglioblastoma, this step would be unnecessary. The data from Yang et al.demonstrate that in human patients, endothelia of the BBB surroundingthe tumor do not overexpress any transporter (including the known Lsystem transporter, A system transporter or ASC transporter) to a degreethat selective anti-angiogenic therapy by BNCT could be practiced. Inagreement with these observations, in their analysis of ¹⁰B-treatedtumor tissue from patients, Imahori et al have not found BPA enrichmentin endothelia, and do not teach to expect ¹⁰B in endothelia.Furthermore, in a systematic study in the Fischer rat glioblastomamodel, Smith et al. (Cancer Res. 61, 8179-8187, 2001) have analyzedserial sections from rat tumors with conventional histology and,alternating, ion microscopy in a direct-imaging secondary ion massspectrometer. The spatial resolution in images generated by thisinstrument is comparable with a high-quality light microscope, allowingimaging of individual nuclei within the tumor architecture and thearchitecture of surrounding tissue. Thus, it should be straightforwardto identify structures resembling blood vessels that enrich ¹⁰B.However, by size, morphology, and tissue architecture, stained cells areunambiguously identifiable as tumor cells, not as microvascularendothelia. Furthermore, while perivascular edema can be assigned bytopology, and be detected as space with low ¹⁰B signal, there is noenrichment of ¹⁰B in the cellular space surrounding or immediatelyadjacent to the edema, while tumor cells are stained distinctively (seeFIG. 1 of Smith et al). These authors observe that the actively growingportion of the tumor infiltrating into surrounding tissue, while clearlydetectable within 2 to 2.5 h after injection of BPA, is not maximallystained in the early phase of the BPA infusion (conventionally set to 1to two hours, see Imahori et al). It is well known in the art thatangiogenic microvessels support tumor growth by their association withthe actively proliferating cell clusters, and that any imaging drugadministered systemically (intravenously or otherwise) will reachinteraction sites such as receptors and transporters in microvascularendothelia first, before interacting with binding sites in a tumor.

Again, the complete absence of BPA signal from vascular tissue eithersurrounding the tumor or penetrating the tumor, while distinctive ¹⁰Bsignals are observed in tumor tissue in the same area of the section, isunambiguous evidence against significant and pharmacologically usefulenrichment of BPA in tumor-associated microvascular endothelia by anymechanism, including hypothetically upregulated protein levels forBPA-specific amino acid transporters, in the therapeutically relevantsituation in vivo.

Last, tumor-associated endothelia are not subject to tumor-specificalterations in gene expression (e.g. they do not acquire drugresistance, or acquire tumor-specific mutations in regulatory genes suchas p53); these differences extend to intracellular signaling and plasmamembrane composition.

Thus, a PET/BNCT strategy targeted at tumor cells and tumor-associatedmicrovascular endothelia is simply not suggested by the data andteachings of the Imahori et al. publications, or the other criticalreferences in the field (see above). Hence, applicant's inventionrepresents the first embodiment of a method to interfere withpathological antigenesis by targeted BNCT, and to monitor the impact ofBNCT in real-time mode.

The possibility of destroying angiogenic tumor-associated endothelia byBCNT, as inherent in the applicant's technology, is particularlyvaluable, because enrichment of BPA is strikingly responsive tofunctional heterogeneities among tumor cell clusters within a giventumor (Imahori et al, 1998), whereas the formation of tumorvascularization does not mirror such functional microheterogeneity.

Conventional ionizing radiation would not be expected to performdestruction of tumor-associated endothelia efficiently in vivo. Whilemicrovascular endothelial cells in vitro may undergo programmed celldeath within 6-10 h after exposure to ionizing radiation (Langley et al,Brit. J. Cancer 75, 666-672, 1997), the apoptotic response may beheterogeneous among tumors of different histologies (Meyn et al, Int J.Radiat. Biol 64, 583-591, 1993), thus causing only incomplete damage tothe vascular supply that is unlikely to destroy the tumor.

Because the gamma radiation component used for imaging in certaincontemplated embodiments of the invention is minimal, and because thethermal neutron ray may be operated in pulse mode, damage to surroundingnon-tumor tissues is anticipated to be minimal in the practice of thepresent invention.

Pharmacological suitable concentration ranges, and modes ofadministration to patients, for radiotracers to be used in PET, SPECT,and BNCT, are well known in the art and may be found in the literaturecited.

Monitoring Gene Expression and Gene Therapy

The invention includes methods and compositions to monitor geneexpression, in particular in the context of gene therapy. The concept ofmapping unknown genes by identifying unique “Expressed Sequence Tags”has been used in the Human Genome Project. Here, we refer to an EST inthe literal sense as an artificially introduced heterologous firstsequence (monitoring sequence) the expression of which is coupled to theexpression of another second sequence (therapeutic sequence).

In one embodiment, the EST may be connected to the therapeutic gene inframe with a sequence encoding a physiological N-terminal membranesorting signal. This approach will lead to overexpressing a therapeuticfusion protein that is physiologically sorted to the membrane (such as asurface receptor, or a regulatory membrane protein like CFTR), bearing adesired biological activity and a unique N-terminal protein tag fordetection. A limitation of this approach is that the protein products ofa gene therapy attempt are not always secreted, and diverting thedesired gene product to a different environment may be harmful andrender the intended therapeutic intervention useless. For detection ofan intracellular protein, it is contemplated to introduce a radiotracerinto the target cell wherein the radiotracer is attached to acomposition comprising a non-agonist ligand to a surface receptor and anintracellular routing moiety delivering the radiotracer into theappropriate cellular microenvironment (a cytoplasmic routing moiety fordetecting a soluble cytoplasmic protein, a nuclear routing sequence fordetecting a nuclear protein, etc.).

The limitations of this strategy are that the tag may interfere withsome aspect of protein function, such as complex formation withallosteric regulators, or complex formation with other proteins thatparticipate upstream or downstream in a signal transduction pathway.Thus, in another embodiment of the invention, indirect detection of atherapeutic gene product is utilized: the EST may be expressed from abicistronic promoter as part of a monitoring gene the product of whichis sorted to the plasma membrane where it is detected by a SPECT-labeledaffinity probe or ligand, whereby the bicistronic promoter will expressthe therapeutic gene with the same efficiency as the monitoring gene.Since the EST-containing protein is not physiologically present inplasma, or on the surface of cells that have not received the genetransfer composition, a signal after the immediate radiotracer washoutperiod is indicative for the presence of the diagnostic EST andtherefore, for successful gene expression.

The heterologous EST may originate from a plant molecule. Van derKrieken and Smit (U.S. Pat. No. 6,242,381) have disclosed chemicallymodified plant growth regulators that are linked to a carrier moleculefor the purpose of increasing the local concentration of auxins in aplant. No application of said modified plant growth regulators outsideof the plant field is contemplated by van der Krieken and Smit.

Currently most preferred embodiment is a pair comprising plantauxin/auxin receptor EST, such as the known ethylene receptor.

As an example for a recognition pair consisting of a plant auxin and aplant auxin receptor, the ligand binding domain of the ethylene receptor(Schaller and Bleecker, Science 270, 1809-1811, 1995), in frame with asequence encoding an N-terminal export signal, may be cloned as EST intoa vector designed to express a therapeutic target gene from abicistronic promoter; the auxin receptor ligand binding domain would beco-expressed at comparable level with the desired therapeutic gene,exported into the plasma membrane, and could be probed by a radiolabeledderivative of ethylene.

Contemplated for gene therapy are all cells within an organism bearing agenetic defect in the unclear portion of the genome, or in themitochondrial portion of the genome. Particularly preferred is thedelivery of the non-viral vector through a suitable and selectivesurface receptor to the cell of a certain cell type that physiologicallyexpresses the gene to be repaired. In case of a secreted protein beingthe product of a deficient gene, the cell targeted with a gene therapyvector need not be the cell that expresses the deficient gene to berepaired.

For administration of a nucleic acid, or a nucleic acid analog, with thepurpose of abrogation or downregulation of the expression of a targetgene encoded in the nuclear or the mitochondrial portion of the genome,all cells bearing a suitable and selective surface receptor arecontemplated wherein the downregulation of a target gene leads to therestoration of a desired cellular function (as a non-limiting example,cell survival of a neuron accumulating toxic proteins, upon interferencewith the expression of toxic proteins), or to the intended eliminationof a pathological cell (e.g. destruction of a tumor cell). Suitableconcentrations for in vivo non-viral gene therapy may vary with gene tobe expressed, localization of the cell target, mode of administration. Apreferred range of 1 to 60 microgram of DNA (suggested by Mixson, U.S.Pat. No. 6,080,728) may have to be modified according to specificconditions, as known in the art.

Alternatively, the heterologous EST may be a hybrid consisting of twoindependent ligand-binding domains (LBDs) comprising the LBD of amutated ecdysone receptor and the LBD of a plant auxin binding protein.No prior art exists for use of ecdysone receptor as EST, or for use ofinsect hormone as PET imaging tracer. As an example, the insect ecdysonereceptor ligand binding domain (Koelle et al, Cell 67, 59, 1991;Christiansen and Kafatos Biochem Biophys Res Comm 193, 1318, 1993;Henrik et al, Nucl. Acid Res. 18, 4143, 1990) may be cloned into avector as described above for the ethylene receptor from the plantArabidopsis thaliana, such that the receptor ligand binding domain isseparated from the promoter by the export domain. Detection would befeasible with a biochemically stable radiolabeled ligand, such asponasteron A which serves as prototype for synthetic ecdysone receptoragonists. Specifically contemplated alternative ligands include thesynthetic ecdysone analogs described by Ravi et al., J. Chem. Inf.Comput. Sci. 41, 1587-1604, 2001. Further contemplated alternativeligands include the bisacylhydrazine analogs described by Smagghe etal., Insect. Biochem. Mol. Biol. 32, 187-192, 2002. Particularlypreferred embodiments are a recognition pair consisting of the ligandbinding domain of a newly discovered G protein-coupled receptorresponsive to ecdysone-like ligands (Park et al., Proc. Natl. Acad. Sci.USA 99, 11423-11428, 2002), or a pair consisting of one out of theecdysone-metabolizing enzymes well known in the art, or catalyticallyimpaired mutants thereof, and ponasterone A. Further contemplatedembodiments include the pair allatostatin/allatostatin receptor (Secheret al, J. Biol. Chem. 276, 47052-47060, 2001), and the pair adipokinetichormone/adipokinetic hormone receptor (Staubli et al., Proc. Natl. Acad.Sci. USA 99, 3446-3451, 2002), and the pair bursicone/bursicone receptor(Baker and Truman, J. Exp. Biol. 205, 2555-2565, 2002).

As an alternative embodiment, a detection pair consisting of juvenilehormone and receptors for juvenile hormone (Palli et al, Proc. Natl.Acad. Sci. USA 87, 796-800, 1990), or as a specifically preferredembodiment, a detection pair consisting of juvenile hormone and juvenilehormone esterase (Hinton and Hammock, Insect Biochem Mol Biol 32, 57-66,2001) is contemplated.

Evans et al. (U.S. Pat. No. 6,333,318) have disclosed the use ofecdysone receptor in conjunction with a co-receptor as a means tocontrol gene expression in heterologous cells and organisms by a smallmolecule ligand.. Evans et al. do not contemplate the use of theecdysone receptor ligand binding domain as an EST to quantify geneexpression by a radiolabeled ecdysone analog. Hogness et al. (U.S. Pat.No. 6,245,531) have disclosed the use of a nucleotide sequence codingfor an ecdysone binding domain. Hogness et al. do not contemplate theuse of mutated ecdysone receptor ligand binding domain as EST, or theuse of a hybrid gene with reduced hybridization specificity.

Functional Imaging of Multidrug Resistance

Functional imaging of multidrug resistance has been performed inpatients with a variety of tumors, using ^(99m)Tc sestamibi [hexakis(2-methoxy-isobutylisonitrile)-technetium(I)] which is a substrate oftwo drug efflux pumps, P-glycoprotein and multidrug-resistance-associated protein. (see for review Hendrikse et al, Eur. J Nucl. Med.26, 283-293, 1999). Certain limitations have been identified in the useof sestamibi, such as unpredictable tumor accumulation(Dimitrakopoulou-Strauss et al, Eur. J Nucl. Med. 22, 434-442, 1995),rapid metabolism in the liver, and problems with the interpretation of apopular parameter in nuclear medicine, the washout rate. The washoutrate from necrotic lung tumors, regardless of drug efflux pump status,may be prone to false-positive signals mimicking particularly highefflux pump activity (Kostakoglu et al, J Nucl Med 39, 228-234, 1998).Because multidrug resistance is a multifactorial phenomenon, it would behighly advantageous to improve current imaging technologies by usingmore defined efflux pump substrates with a higher specificity forindividual components of multidrug resistance pathways. Furthermore, thechemical characteristics of sestamibi and related compounds are that ofa lipophilic cation which is known to cross membranes depending on thedegree of membrane polarization, and to accumulate in mitochondria.Thus, uptake and redistribution of sestamibi are controlled by complexparameters that are likely to vary substantially among patients andtumor types. It is apparent that the invention presented herein placesthe uptake of an imaging agent under control of a measurable parameter,the density of a surface receptor such as SSR-2 receptor, and accountsfor enrichment in a subcellular environment that can be pre-determinedby the choice of the routing moiety. Measuring activity of an individualtype of drug efflux pump before, concurrent with, and afteradministration of a BAM reducing the expression and activity of genesencoding proteins regulating drug efflux is a further improvement overcurrently existing methods that attempt to monitor the drug efflux pumpfunctionality. The decreased clearance of the radioligand from the tumorcan be monitored by measuring the PET or SPECT signal intensity overtime in the ROI, and can directly be correlated with the kinetics ofreduced gene expression of the mdr gene of interest. Furthermore, forthe purpose of reversing the multidrug resistance phenotype of thetumor, the invention is suitable to deliver into the cytoplasm blockingagents for drug efflux pumps of a size that is unlikely to permit rapidclearance by any member of the drug efflux pump families, It ispredicted that the targeted/routed application of efflux pump blockerswill improve the chances for a response to conventional chemotherapy,e.g. by mitoxantrone, beyond the disappointing level seen in patientswho had received oral verapamil (Hendrick et al, Ann. Oncol. 2, 71-72,1991) or cyclosporin A as adjuvants (Rodenburg et al, Ann. Oncol. 2,305-306, 1991)

Functional Imaging of Biological Processes in Mitochondria

Steliou (U.S. Pat. No. 6,316,652) has disclosed a targeted drug agentconsisting of a highly soluble cisplatin derivative, a lipophilic cationcomposition, and a second mitochondrial targetor compound, such ascarnitine or a carnitine analog, that render the targeted drug agentsusceptible to transport through the mitochondrial membrane via theL-carnitine acylcarnitine translocase system. The targeted drug agent iscontemplated for therapeutic and imaging applications. U.S. Pat. No.6,316,652 lacks the additional selection principle for a surfacereceptor with selectivity for pathological cells that is embodied inapplicant's invention. Owing to the lack of selectivity forpathologically proliferating microvascular endothelia, the use forantiangiogenic therapy, or for imaging of pathological angiogenesis, isnot contemplated. No experimental evidence is presented whether thecomposition will have significant selectivity for tumor mitochondria invivo, while showing minimal accumulation into mitochondria ofnon-tumorous cells. Wallace and Brown (U.S. Pat. No. 5,670,320)contemplate encoding the sequence of a mitochondrial targeting peptidein a gene therapy vector, to facilitate import of a replacement peptidefor the mitochondrial ND6 protein deficient in patients with Leber'sHereditary Optic Neuropathy. Imaging of mitochondrial gene defect(s) byPET or SPECT is not disclosed. Muzyczka et al. (U.S. Pat. No. 6,020,192)describe the use of targeting green fluorescent protein and its variantsto mitochondria by means of an N-terminal targeting sequence. An 18merpeptide is given as example. Bandman et al. (U.S. Pat. No. 6,432,915)disclose the sequence of a human mitochondrial chaperone proteinHMt-GrpE and contemplate the use of portions and fragments intherapeutic and diagnostic compositions.

Inherited and acquired mitochondrial dysfunction has been recognized asan important mechanistic principle in diseases affecting the liver andother organ systems (Treem and Sokol, Semin. Liver Dis. 18, 237-253,1998). Hepatic failure or chronic liver dysfunction can develop into alife-threatening condition; however, diagnosis of mitochondrialdysfunction may be difficult with currently available tools. It is thusdesirable to have expanded imaging capabilities of intact and healthymitochondria, and of real-time measurements of heteroplamsicmitochondria fraction vs. homoplasmic fraction. Prior art based onlipophiliccations as targeting elements carries the risk of introducinga bias into imaging signals, in that mitochondrial access of the imagingdrug is facilitated if the mitochondria are functionally impaired orpart of a diseased cell type. Moreover, prior art using targetingpeptides did not contemplate a combination of a targeting moiety, arouting moiety directed to a substrate for a mitochondrial pathway and aradionuclide for PET or SPECT imaging

In another preferred embodiment of the invention, the routing sequencemay be a mitochondrial import peptide modeled after the mutatedmitochondrial aldehyde dehydrogenase signal peptide LRAALSTARRLSRLL (SEQID NO: 1) (Thornton et al, J. Biol. Chem 268, 19906-19914, 1993) areused as a part of such a DDM. Alternative embodiments include thepresequence peptide of mammalian hsp60 MLRLPTVLRQMRPVSRALAPHLTRAYC (SEQID NO: 2) (Haucke et al, J Biol. Chem 270, 5565-5570, 1995), thecytochrome oxidase subunit IV LSLRQSIRFFKPATRTLCSSR(SEQ ID NO: 3) (Endoet al, J Biochem. 106, 396-400, 1989), and sequences and sequenceelements reviewed in Hartl and Neupert, Science 247, 930-938 (1990) andPfanner et al., Annu Rev. Cell Dev. biol. 13, 25-51, 1997). Anotherpreferred routing sequence isLys-Lys-Arg-Lys-Leu-Ile-Glu-Glu-Asn-Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ IDNO: 4). A presently preferred targeting sequence isDPhe-Cys-Phe-DTrp-Lys-Thr-Apa-Cys-Thr (cyclized by Cys-Cys bridge).

For the purpose of detecting mitochondrial deficiencies with thepotential to develop into life-threatening dysfunctions (e.g. uremia andhyperammonemia), a preferred embodiment of the DDM may include a BAMwhich is a substrate of any of the enzymes on the respiratory chain. Analternative embodiment is as substrate for the very-long chain acyl-CoAdehydrogenase EC 1.3.99.13 linked to sudden death in childhood (Strausset al, Proc Natl Acad Sci USA 92, 10496 10500, 1995); another embodimentis one that is aimed at diagnosis and treatment of congenital urea cycledisorders resulting in acute hyperammonemia (most recently reviewed inFelipo and Butterworth, Neurochem Int. 40, 487-491, 2002) and comprisesa substrate of alpha ketoglutarate dehydrogenase and an antagonist ofthe NMDA receptor.

Early non-invasive diagnosis of beginning organ failure with highlypredictive value is a much-needed improvement in intensive caremedicine.

Improvements over Somatostatin-Agonists with Limited ReceptorSelectivity

It should further be pointed out that toxicity has been observed withradiolabeled octreotide (Nucl. Med. Comm. 21, 97-102, 2000) as well aswith unlabeled octreotide. Generally, octreotide causes delay incarbohydrate absorption and glucose production by the liver. While mostpatients with acromegalia treated with octreotide seem to have noadverse effects on glucose tolerance, in some cases, hypoglycemia hasbeen found, which was explained by octreotide causing an imbalancebetween insulin-mediated and growth hormone-mediated blood glucoseregulation (Popovic et al., Digestion 54(Suppl 1), 104-106, 1993). Up to20% of patients develop diarrhea, and in one patient collectivegastritis with subsequent decline in vitamin B12 absorption has beenreported Plockiner et al, J. Clin. Endocrinol. Metab. 71, 1658-1662,1990). By far the most serious side effect of octreotide is thecombination of reduction in bile flow in humans (Gullo et al., Dig. Dis.Sci. 31, 1345-1350, 1986; Magnusson et al., Gastroenterology 96,206-212, 1989) and gall bladder contractility. The reduction of gallbladder contractility in response to a meal was connected by severalgroups of investigators to the reduction, if not inhibition, ofcholecystokinine levels in the plasma of octreotide-treated patients(see e.g. van Lissum et al., J. Clin. Endocrinol. Metab. 69, 557-562,1989). The increased incidence of gallstones in response to octreotidetreatment was reviewed by Dowling et al., Digestion 54(Suppl 1),107-120, 1993. It was concluded that the timing of meals andsubcutaneous octreotide injections necessary for reducing the gallbladder side effects was impractical and would defeat the whole purposeof octreotide treatment in acromegalia. The same conclusion holds forcancer patients where an infusion of octreotide might be necessary toachieve the higher doses needed; under conditions of infusion, Dowlinget al reported a continuous inhibition of the cholecystokinine response.There is an urgently felt need to improve somatostatin analogs byreducing the cholestatic and metabolic side effects. Recently, a novelsynthetic cyclic analog of somatostatin, P829, (Vallabhajosula et al.,J. Nucl. Med. 37, 1016-1022, 1996) has been introduced as depreotideinto clinical evaluation for 99m-Tc-SPECT scanning of lung cancer andneuroendocrine tumors. In pre-clinical investigation, 99m-Tc-depreotidedemonstrated high affinity binding to the SSR subtypes 3, 5, and 2(Virgolini et al, Cancer Res. 58, 1850-1859, 1998). However, the freepeptide has an apparent affinity to the ssr subtypes present in ratpancreas carcinoma membranes that is one order of magnitude lower thanthe affinity of octreotide (Vallahabjosula et al.), whereas theradiolabeled peptide shows higher affinity. It is a concern thatmodifications of the radiotracer in vivo may alter receptor affinitysubstantially, introduce radiotracer populations with differentaffinities, and generate ambiguity in image interpretation. In contrastto findings with octreotide, discrepancies in the scintigraphic resultswere seen in one third of neuroendocrine tumor patients treated withlanreotide (Virgolini et al, Q J Nucl Med. 45, 153-159, 2001) whichresembles depreotide in having similar affinities to receptor subtypes.In comparison to octreotide, receptor selectivity of depreotide is notnarrowed, but widened, opening depreotide to the same risk of seriousside effects that has been recognized as causing a need for furtherimprovement. It should be emphasized that none of the references ondepreotide teaches the use of non-agonists in combination with anintracellular routing moiety.

The technology of the applicant improves on the prior art by avoidingthe known side effects of activating the SSR-2, and setting a novelparadigm of functional imaging of a wide array of intracellularprocesses, by delivering radiotracers to pre-determined subcellularmicroenvironments selectively in cell populations characterized byexpressing a receptor of interest. Furthermore, the technology extendsthe biological half-life of the imaging drug at the desired target site,improving safety by reducing the amount of radioactivity needed,improving imaging performance by permitting repeated and multiple scansfrom the same dose of radiotracer administered. The technology expandsdiagnostic options for somatostatin-based analogs to tumor angiogenesis,macular degeneration, and a variety of other angiogenic disorders.Applicant has come to the conclusion that the use of non-agonists,conventionally considered worthless in therapy, will overcome theserious limitations of octreotide and other SSR agonists disclosed inprior art Applicant has further demonstrated that the delivery of anon-agonist ligand is feasible in vivo and in vitro. While it is knownin the art that somatostatin does not enter the nucleus, a compositioncomprising a non-agonist ligand and a nuclear routing moiety has beenshown by the applicant to enter the nucleus. Therefore, somatostatinreceptor-dependent targeting could be rendered useful forpharmacological modulation of processes in the nucleus.

An example of one DDM with a potential for anti-apoptotic gene therapyincludes a selective SSTR2-ligandPro-cyclo[Cys-Lys-Asn-Asu-Phe-D-betaMeTrp-Lys-Abu-Tyr-Ser-Ser-Cys]-Lysthat is designed to deliver the sequence to a specific group of targetcells, such as microvascular endothelia in Alzheimer's Disease. TheSSTR2-selective ligand is connected to an N-terminal nuclear routingsignal Arg-Arg-Ser-Met-Lys-Arg-Lys (SEQ ID NO: 31) contiguous to anintegrated linkage enabler Pro-Orn-Pro-Orn-Pro-Orn-Asp-NH₂ (SEQ ID NO:32) and a second modified C-terminal nuclear routing signalPro-Lys-Lys-Lys-Arg-Lys-Val-Asp (SEQ ID NO: 33) that branches off thesecond Orn residue of the linkage enabler. Such a linkage enablerfurther comprises an aspartic acid-aminoguanine substituent to which the5′ end of the desired vector DNA is covalently coupled. The 3′ end ofthe DNA terminates in another aminoguanine which is coupled toPro-Orn-Pro-Orn (SEQ ID NO: 34) connected to the carboxy terminus of thesecond nuclear routing signal. The DNA sequence comprises a minimal vWFpromoter that is specific for endothelial gene expression followed bythe coding sequence for heme oxygenase I.

The facility of the invention has been demonstrated by the successfultargeting of tumor cells in culture by SSR-2 non-agonist ligands linkedto a nuclear routing moiety and a marker dye. Such has shown enrichmentof that marker dye in the nucleus, by a factor of 30 or more, 48 h afterapplication of the compound. Moreover, confocal imaging has shown veryhigh levels SSR-2 expression in proliferating microvascular endotheliasurrounding human glioblastoma, illustrating the usefulness of a DDMincorporating an SSR-2 ligand. In addition, confocal image of successfultargeting of SSR-non-agonists to tumor and endothelial nuclei in axenograft mouse model for advanced prostate cancer. When tested in axenograft mouse model for advanced prostate cancer, only nuclei of tumorcells and angiogenic endothelial cells were found labeled 24 h afterapplication of the SSR-2 non-agonist compound.

It can thus be seen that, the current invention overcomes thelimitations in enrichment and target selectivity presented by currentBCNT drugs such as BPA, and by further entailing PET(SPECT)capabilities, provides a means to validate and quantify the desiredlocalization and concentration of the BCNT drug before treatment, and tomonitor the loss of tumor cells (e.g. scored as signal reduction due toloss of detectable tumor nuclei) immediately after treatment.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventor, it should be understood that various changes and modificationsas would be obvious to one having the ordinary skill in this art may bemade without departing from the scope of the invention which is setforth in the claims appended hereto. The disclosures of all U.S. patentsand publications mentioned hereinbefore are expressly incorporated byreference.

1. A drug delivery molecule which comprises a. a bioactive molecule(BAM) which includes nucleic acid designed to effect gene therapy and anexpressed sequence tag (EST) which comprises nucleic acid encoding aligand-binding element from a plant auxin binding protein, or an insecthormone binding protein, linked in-frame to a membrane-sorting element,b. a targeting moiety that does not activate a targeted receptor towhich it links, and c. a routing sequence for causing delivery to asubcellular compartment in a cell having said targeted receptor.
 2. Themolecule of claim 1 wherein said targeted receptor is a somatostatinType 2 receptor.
 3. The molecule of claim 2 wherein the BAM includes aboron isotope that will capture a slow neutron, decay and releasecell-destructive radiation.
 4. The molecule of claim 1 wherein therouting sequence is a mitochondrial import moiety.
 5. The molecule ofclaim 4 wherein the BAM is cleavable by mitochondrial translocase and asubstrate of a metabolic pathway designed for gene repair localized inmitochondria or is designed for gene repair.
 6. The molecule of claim 1wherein the routing sequence is selected from the group consisting ofSEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4.
 7. A non-viralgene delivery vector comprising the molecule of claim
 1. 8. A method ofdelivering the vector of claim 7 to a cell or living organism, includinga human patient, and monitoring the expression of the therapeuticnucleic acid, which method comprises first administering the vector tothe living organism; then, after the first delivery, administering aligand to a plant auxin binding protein or to an insect hormone bindingprotein, which ligand is combined with a label useful for imaging byPositron Emission Tomography (PET), Magnetic Resonance Imaging (MRI) orsingle Photon Emission Computed Tomography (SPECT); and then detectingand quantifying the presence of the EST by scanning and processing PET,MRI or SPECT signals emitted from the label.
 9. A drug delivery moleculewhich comprises a. a bioactive molecule (BAM) that includes an expressedsequence tag (EST) encoding a ligand-binding element from a plant auxinbinding protein, or from an insect hormone binding protein, linked to anucleic acid sequence complementary to a gene desired to be reduced inexpression, b. a targeting moiety that does not activate a targetedreceptor to which it links, and c. a routing sequence for causingdelivery to a subcellular compartment in a cell having said targetedreceptor.
 10. The molecule of claim 9 wherein the targeted receptor is asomatostatin Type 2 receptor.
 11. The molecule of claim 9 wherein therouting sequence is a mitochondrial import moiety.
 12. The molecule ofclaim 9 wherein the routing sequence is selected from the groupconsisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4.13. A non-viral gene delivery vector comprising the molecule of claim 9.14. A method of delivering the non-viral vector of claim 13 to a cell orliving organism, including a human patient, and monitoring the reductionin expression of the desired gene, which comprises first administeringthe vector to the cell or living organism, then administering a ligandto a plant auxin binding protein or to an insect hormone bindingprotein, which ligand is combined with a label useful for imaging byPositron Emission Tomography (PET), Magnetic Resonance Imaging (MRI) orSingle Photon Emission Computed Tomography (SPECT), and then detectingand quantifying the time-dependent reduction of the EST by way ofscanning and processing PET, MRI or SPECT signals emitted from thelabel.
 15. A drug delivery molecule which comprises a. a bioactivemolecule (BAM), b. a targeting moiety that does not activate a targetedreceptor to which it links, c. a routing sequence for causing deliveryto a subcellular compartment in a cell having said receptor and d. alabel useful for imaging by Positron Emission Tomography (PET), MagneticResonance Imaging (MRI) or Single Photon Emission Computed Tomography(SPECT).
 16. The molecule of claim 15 wherein the targeted receptor is asomatostatin Type 2 receptor.
 17. The molecule of claim 15 wherein thelabel includes one out the group of ¹⁸F, ¹¹C, ¹²³I, ¹²⁴I, ⁶⁴Cu, ^(99m)Tcand ¹¹¹In.
 18. The molecule of claim 15 wherein the BAM can be locallyactivated to destroy cells into which said drug delivery molecule hasbeen translocated.
 19. The molecule of claim 15 wherein the BAM includesan apoptosis-inducer.
 20. The molecule of claim 19 wherein theapoptosis-inducer is tissue-specific.